Immunotherapy with binding agents

ABSTRACT

Binding agents that modulate the immune response are disclosed. The binding agents may include soluble receptors, polypeptides, and/or antibodies. Also disclosed are methods of using the binding agents for the treatment of diseases such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/088,888, filed Apr. 1, 2016, which is a divisional of U.S.application Ser. No. 14/096,510, filed Dec. 4, 2013, now U.S. Pat. No.9,327,014, which claims priority benefit of U.S. Provisional ApplicationNo. 61/733,177, filed Dec. 4, 2012 and U.S. Provisional Application No.61/789,268, filed Mar. 15, 2013 each of which is hereby incorporated byreference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:2293_0990004_SeqListing.TXT; Size: 118,202 bytes; and Date of Creation:Oct. 30, 2017) filed herewith is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention generally relates to agents that modulate the immuneresponse, such as soluble receptors, antibodies, and small molecules, aswell as to methods of using the agents for the treatment of diseasessuch as cancer.

BACKGROUND OF THE INVENTION

The basis for immunotherapy is the manipulation of the immune system,including both innate immune responses and adaptive immune responses.The aim of immunotherapy is to treat diseases by controlling the immuneresponse to a “foreign agent”, for example a pathogen or a tumor cell.This may include methods to induce or enhance specific immune responsesor to inhibit or reduce specific immune responses. The immune system isa highly complex system made up of a great number of cell types,including, T-cells, B-cells, natural killer cells, antigen-presentingcells, dendritic cells, monocytes, and macrophages. These cells possesscomplex and subtle systems for controlling their interactions, includingutilizing numerous receptors and soluble factors for the process. Thecells utilize both activating and inhibitory mechanisms to keepresponses in check and not allow negative consequences of anuncontrolled immune response (e.g., autoimmune diseases).

The concept of cancer immunosurveillance is based on the theory that theimmune system can recognize tumor cells, mount an immune response, andsuppress the development and/or progression of a tumor. However, it isclear that many cancerous cells have developed mechanisms to evade theimmune system allowing the uninhibited growth of tumors. Cancerimmunotherapy focuses on the development of agents that can activateand/or boost the immune system to achieve a more effective response tokilling tumor cells and inhibiting tumor growth.

BRIEF SUMMARY OF THE INVENTION

The present invention provides binding agents, such as solublereceptors, polypeptides, antibodies, and small molecules that modulatethe immune response. The invention also provides compositions, such aspharmaceutical compositions, comprising the binding agents. Theinvention further provides methods of administering the binding agentsto a subject in need thereof.

In one aspect, the invention provides a binding agent that specificallybinds the extracellular domain of human TIGIT. As used herein, a“binding agent” includes but is not limited to, a soluble receptor, apolypeptide, an antibody, a small molecule, and combinations thereof. Insome embodiments, the binding agent comprises a soluble receptor. Insome embodiments, the binding agent comprises a soluble receptorcomprising a poliovirus receptor (PVR) variant. In some embodiments, thebinding agent is a soluble receptor comprising a poliovirus receptor(PVR) variant. In some embodiments, the binding agent comprises asoluble receptor comprising a PVR variant, wherein the PVR variantcomprises one or more amino acid substitutions as compared to wild-typePVR. In some embodiments, the binding agent comprises a soluble receptorcomprising a PVR variant which specifically binds the extracellulardomain of human TIGIT and does not bind or binds weakly to theextracellular domain of human CD226. In some embodiments, the bindingagent comprises a soluble receptor comprising a PVR variant whichspecifically binds the extracellular domain of human TIGIT and alsobinds the extracellular domain of human CD96. In some embodiments, thebinding agent comprises a soluble receptor comprising a PVR variant thatspecifically binds the extracellular domain of human TIGIT and theextracellular domain of human CD96, but does not bind or binds weakly tothe extracellular domain of human CD226. In some embodiments, thebinding agent comprises a soluble receptor comprising a PVR variant thatspecifically binds the extracellular domain of human TIGIT and hasreduced binding to the extracellular domain of human CD226 as comparedto wild-type PVR. In some embodiments, the binding agent comprises asoluble receptor comprising a PVR variant which specifically binds theextracellular domain of human TIGIT and the extracellular domain ofhuman CD96, but has reduced binding to the extracellular domain of humanCD226 as compared to wild type PVR.

In some embodiments, the PVR variant comprises one or moreimmunoglobulin (Ig)-like domains of human PVR. In some embodiments, thePVR variant comprises an N-terminal IgV domain of human PVR. In someembodiments, the PVR variant comprises an N-terminal IgV domain of humanPVR, wherein the IgV domain comprises one or more amino acidsubstitutions as compared to wild-type PVR. In some embodiments, the PVRvariant consists essentially of an N-terminal IgV domain of human PVR,wherein the IgV domain comprises one or more amino acid substitutions ascompared to wild-type PVR. The amino acid sequence of human PVR is knownin the art and is included herein as SEQ ID NO:1. In some embodiments,the PVR variant comprises substitutions in one or more amino acidscorresponding to amino acids 40-143 of wild-type PVR. In someembodiments, the PVR variant comprises substitutions in one or moreamino acids corresponding to amino acids 60-90 and/or amino acids125-133 of wild-type PVR. In some embodiments, the PVR variant comprisessubstitutions in one or more amino acids corresponding to amino acids465, 67, 72, 73, 74, 81, 82, 84, and 85 of wild-type PVR. In someembodiments, the PVR variant comprises an amino acid substitutioncorresponding to amino acid 72 of wild-type PVR. In some embodiments,the PVR variant comprises an amino acid substitution corresponding toamino acid 82 of wild-type PVR. In some embodiments, the PVR variantcomprises amino acid substitutions corresponding to amino acid 72 andamino acid 82 of wild-type PVR. In some embodiments, the PVR variantcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

In another aspect, the invention provides a polypeptide comprising oneor more Ig-like domains of human PVR, wherein the one or more Ig-likedomains comprise substitutions in one or more amino acids as compared towild-type PVR. In some embodiments, the polypeptide specifically bindsthe extracellular domain of human TIGIT and does not bind or bindsweakly to the extracellular domain of human CD226. In some embodiments,the polypeptide comprises an N-terminal IgV domain of human PVR. In someembodiments, the polypeptide comprises an IgV domain of PVR thatcomprises an N-terminal IgV domain of human PVR, wherein the IgV domaincomprises one or more amino acid substitutions as compared to wild-typePVR. In some embodiments, the polypeptide comprises an IgV domain of PVRthat consists essentially of an N-terminal IgV domain of human PVR,wherein the IgV domain comprises one or more amino acid substitutions ascompared to wild-type PVR. In some embodiments, a polypeptide comprisesa PVR variant, wherein the PVR variant comprises one or more amino acidsubstitutions as compared to wild-type PVR. In some embodiments,polypeptide comprises a PVR variant that specifically binds theextracellular domain of human TIGIT and does not bind or binds weakly tothe extracellular domain of human CD226. In some embodiments, thepolypeptide also binds the extracellular domain of human CD96. In someembodiments, the polypeptide comprises a PVR variant, wherein the PVRvariant comprises substitutions in one or more amino acids correspondingto amino acids 40-143 of wild-type PVR. In some embodiments, thepolypeptide comprises a PVR variant, wherein the PVR variant comprisessubstitutions in one or more amino acids corresponding to amino acids60-90 and/or amino acids 125-133 of wild-type PVR. In some embodiments,the polypeptide comprises a PVR variant, wherein the PVR variantcomprises substitutions in one or more amino acids corresponding toamino acids 465, 67, 72, 73, 74, 81, 82, 84, and 85 of wild-type PVR. Insome embodiments, the polypeptide comprises a PVR variant, wherein thePVR variant comprises an amino acid substitution corresponding to aminoacid 72 of wild-type PVR. In some embodiments, the polypeptide comprisesa PVR variant, wherein the PVR variant comprises an amino acidsubstitution corresponding to amino acid 82 of wild-type PVR. In someembodiments, the polypeptide comprises a PVR variant, wherein the PVRvariant comprises amino acid substitutions corresponding to amino acid72 and amino acid 82 of wild-type PVR. In some embodiments, thepolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ IDNO:21.

In another aspect, the invention provides a TIGIT-binding agentcomprising one or more Ig-like domains of a variant human PVR, whereinthe one or more Ig-like domains of PVR comprise one or more amino acidsubstitutions as compared to wild-type PVR. In another aspect, theinvention provides a TIGIT-binding agent comprising one or more Ig-likedomains of a variant human PVR, wherein the one or more Ig-like domainsof PVR comprise one or more substitutions in amino acids correspondingto amino acids 65, 67, 72, 73, 74, 81, 82, 84, or 85 of wild-type PVR.In some embodiments, the TIGIT-binding agent comprises one or moreIg-like domains of a variant human PVRL2, wherein the one or moreIg-like domains of PVRL2 comprise one or more amino acid substitutionsas compared to wild-type PVRL2. In some embodiments, the TIGIT-bindingagent comprises one or more Ig-like domains of a variant human PVRL3,wherein the one or more Ig-like domains of PVRL3 comprise one or moreamino acid substitutions as compared to wild-type PVRL3. In someembodiments, the TIGIT-binding agent comprises one or more Ig-likedomains of a variant human PVRL4, wherein the one or more Ig-likedomains of PVRL4 comprise one or more amino acid substitutions ascompared to wild-type PVRL4.

In some embodiments of each of the aforementioned aspects andembodiments, as well as other aspects and embodiments described herein,the binding agent comprises a non-PVR polypeptide. In some embodiments,the PVR variant is linked to a non-PVR polypeptide. In some embodiments,the PVR variant is directly linked to a non-PVR polypeptide. In someembodiments, the PVR variant is linked to a non-PVR polypeptide with apeptide linker. In some embodiments, the non-PVR polypeptide comprises ahuman Fc region. In some embodiments, the non-PVR polypeptide consistsessentially of a human Fc region. In some embodiments, the non-PVRpolypeptide consists of a human Fc region. In some embodiments, thehuman Fc region is selected from the group consisting of SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,and SEQ ID NO:48.

In some embodiments of each of the aforementioned aspects andembodiments, as well as other aspects and embodiments described herein,the binding agent is monovalent. In some embodiments, the binding agentis bivalent. In some embodiments, the binding agent is monospecific. Insome embodiments, the binding agent is bispecific.

In some embodiments of each of the aforementioned aspects andembodiments, as well as other aspects and embodiments described herein,the binding agent is a heteromultimeric agent. In some embodiments, thebinding agent is a heterodimeric agent. In some embodiments, theheterodimeric agent comprises a first polypeptide that binds TIGIT and asecond polypeptide that binds a second target. In some embodiments, theheterodimeric agent comprises a first polypeptide that binds TIGIT and asecond polypeptide that comprises an immune response stimulating agent.In some embodiments, the heterodimeric agent comprises a firstpolypeptide comprising a PVR variant described herein and a secondpolypeptide comprising an immune response stimulating agent. In someembodiments, the immune response stimulating agent may be, but is notlimited to, granulocyte-macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF), granulocyte colonystimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12(IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 15(IL-15), CD80, CD86, anti-CD3 antibody, anti-CTLA-4 antibody, and/oranti-CD28 antibody. In some embodiments, the heterodimeric agentcomprises two polypeptides, wherein each polypeptide comprises a humanIgG2 CH3 domain, and wherein the amino acids at positions correspondingto positions 249 and 288 of SEQ ID NO:40 of the first IgG2 CH3 domainare replaced with glutamate or aspartate, and wherein the amino acids atpositions corresponding to positions 236 and 278 of SEQ ID NO:40 of thesecond IgG2 CH3 domain are replaced with lysine.

In some embodiments of each of the aforementioned aspects andembodiments, as well as other aspects and embodiments described herein,the binding agent increases cell-mediated immunity. In some embodiments,the binding agent increases T-cell activity. In some embodiments, thebinding agent increases cytolytic T-cell (CTL) activity. In someembodiments, the binding agent increases natural killer (NK) cellactivity. In some embodiments, the binding agent is an antagonist ofTIGIT-mediated signaling. In some embodiments, the binding agent is anantagonist of CD96-mediated signaling. In some embodiments, the bindingagent inhibits TIGIT signaling. In some embodiments, the binding agentinhibits CD96 signaling. In some embodiments, the binding agent inhibitsTIGIT signaling and CD96 signaling. In some embodiments, the bindingagent increases CD226 signaling. In some embodiments, the binding agentinhibits TIGIT signaling, inhibits CD96 signaling, but does not inhibitCD226 signaling. In some embodiments, the binding agent inhibits TIGITsignaling, inhibits CD96 signaling, and increases CD226 signaling. Insome embodiments, the binding agent inhibits or blocks the interactionbetween PVR and TIGIT. In some embodiments, the binding agent inhibitsor blocks the interaction between PVR and TIGIT and the interactionbetween PVR and CD96. In some embodiments, the binding agent inhibits orblocks the interaction between PVR and TIGIT, inhibits or blocks theinteraction between PVR and CD96, but does not inhibit or block theinteraction between PVR and CD226. In some embodiments, the bindingagent inhibits or blocks the interaction between PVRL2 and TIGIT. Insome embodiments, the binding agent inhibits or blocks the interactionbetween PVRL3 and TIGIT. In some embodiments, the binding agent inhibitsor blocks the interaction between PVRL4 and TIGIT.

In another aspect, the invention provides pharmaceutical compositionscomprising a soluble receptor, an antibody, a polypeptide, or a bindingagent described herein and a pharmaceutically acceptable carrier.Methods of treating cancer and/or inhibiting tumor growth in a subject(e.g., a human) comprising administering to the subject an effectiveamount of a composition comprising the binding agents described hereinare also provided.

In certain embodiments of each of the aforementioned aspects, as well asother aspects and/or embodiments described elsewhere herein, the solublereceptor, the antibody, the polypeptide, or the binding agent isisolated. In certain embodiments, the soluble receptor, the polypeptide,or the binding agent is substantially pure.

In another aspect, the invention provides polynucleotides comprising apolynucleotide that encodes a soluble receptor, an antibody, apolypeptide, or a binding agent described herein. In some embodiments,the polynucleotide is isolated. In some embodiments, the inventionfurther provides vectors that comprise the polynucleotides, as well ascells that comprise the vectors and/or the polynucleotides. In someembodiments, the invention also provides cells comprising or producing asoluble receptor, an antibody, a polypeptide, or a binding agentdescribed herein. In some embodiments, the cell is a monoclonal cellline. In some embodiments, the cell is a prokaryotic cell. In someembodiments, the cell is an eukaryotic cell.

In another aspect, the invention provides methods of modulating theimmune response of a subject. In some embodiments, the inventionprovides a method of increasing an immune response in a subjectcomprising administering to the subject a therapeutically effectiveamount of a binding agent described herein. In some embodiments, theinvention provides a method of activating an immune response in asubject comprising administering to the subject a therapeuticallyeffective amount of a binding agent described herein. In someembodiments, the immune response is to an antigenic stimulation. In someembodiments, the antigenic stimulation is a tumor or a tumor cell. Insome embodiments, the antigenic stimulation is a pathogen. In someembodiments, the antigenic stimulation is a virus. In some embodiments,the antigenic stimulation is a virally-infected cell. In someembodiments, the invention provides a method of increasing the activityof immune cells. In some embodiments, the invention provides a method ofincreasing the activity of CD226-positive cells comprising contactingthe cells with an effective amount of a binding agent described herein.In some embodiments, the CD226-positive cells are T-cells, NK cells,monocytes, macrophages, and/or B-cells. In some embodiments, theinvention provides a method of increasing the activity of NK cells in asubject comprising administering to the subject a therapeuticallyeffective amount of a binding agent described herein. In someembodiments, the invention provides a method of increasing the activityof T-cells in a subject comprising administering to the subject atherapeutically effective amount of a binding agent described herein. Insome embodiments, the invention provides a method of increasing theactivation of T-cells and/or NK cells in a subject comprisingadministering to the subject a therapeutically effective amount of abinding agent described herein. In some embodiments, the inventionprovides a method of increasing the T-cell response in a subjectcomprising administering to the subject a therapeutically effectiveamount of a binding agent described herein. In some embodiments, theinvention provides a method of increasing the activity of CTLs in asubject comprising administering to the subject a therapeuticallyeffective amount of a binding agent described herein. In someembodiments, the invention provides a method of increasing an immuneresponse in a subject comprising administering to the subject atherapeutically effective amount of a soluble receptor comprising a PVRvariant, wherein the soluble receptor (i) inhibits the interactionbetween TIGIT and PVR and (ii) inhibits the interaction between CD96 andPVR. In some embodiments, the invention provides a method of increasingan immune response in a subject comprising administering to the subjecta therapeutically effective amount of a soluble receptor comprising aPVR variant, wherein the soluble receptor (i) inhibits the interactionbetween TIGIT and PVR, (ii) inhibits the interaction between CD96 andPVR, and (iii) does not inhibit the interaction between CD226 and PVR.

In another aspect, the invention provides methods of inhibiting tumorgrowth in a subject comprising administering to the subject atherapeutically effective amount of a binding agent described herein. Insome embodiments, the invention provides a method of inhibiting tumorgrowth comprising contacting cells with an effective amount of a solublereceptor comprising a PVR variant. In some embodiments, the inventionprovides a method of inhibiting tumor growth comprising contacting cellswith an effective amount of a soluble receptor comprising a PVR variant,wherein the soluble receptor (i) inhibits the interaction between TIGITand PVR, (ii) inhibits the interaction between CD96 and PVR, and (iii)does not inhibit the interaction between CD226 and PVR. In someembodiments, the invention provides a method of inhibiting tumor growthin a subject comprising administering to the subject a therapeuticallyeffective amount of a binding agent described herein. In someembodiments, the invention provides a method of inhibiting tumor growthin a subject comprising administering to the subject a therapeuticallyeffective amount of a binding agent described herein, wherein thebinding agent specifically binds the extracellular domain of human TIGITand inhibits TIGIT signaling and does not inhibit CD226 signaling. Insome embodiments, the invention provides a method of inhibiting tumorgrowth in a human subject comprising determining if the tumor has anelevated expression level of PVR as compared to a reference sample or apre-determined level of PVR, and administering to the subject atherapeutically effective amount of a binding agent described herein.

In another aspect, the invention provides methods of treating cancer ina subject comprising administering to the subject a therapeuticallyeffective amount of a binding agent described herein.

In some embodiments of each of the aforementioned aspects andembodiments, as well as other aspects and embodiments described herein,the methods comprise administering to the subject an immune responsestimulating agent. In some embodiments, the immune response stimulatingagent is selected from a group consisting of, but not limited to,GM-CSF, M-CSF, G-CSF, IL-3, IL-12, IL-15, IL-1, IL-2, CD80, CD86,anti-CD3 antibodies, anti-CTLA-4 antibodies, and anti-CD28 antibodies.

In another aspect, the invention provides methods of selecting a humansubject for treatment with a binding agent described herein comprising,determining if the subject has a tumor that has an elevated expressionlevel of PVR as compared to a reference sample or a pre-determined levelof PVR, wherein if the tumor has an elevated expression level of PVR thesubject is selected for treatment.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but also eachmember of the group individually and all possible subgroups of the maingroup, and also the main group absent one or more of the group members.The present invention also envisages the explicit exclusion of one ormore of any of the group members in the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Alignment of the N-terminal Ig domains of members of the PVRfamily.

FIG. 2. Diagram of PVR family member, membrane-bound decoy receptor, andsoluble receptor.

FIGS. 3A, 3B, and 3C. FACS analysis of binding interactions between PVRfamily members. (3A) HEK-293T cells were transiently transfected with acDNA expression vector encoding PVR-CD4TM-GFP, PVRL1-CD4TM-GFP,PVRL2-CD4TM-GFP, PVRL3-CD4TM-GFP, or PVRL4-CD4TM-GFP and thensubsequently mixed with soluble CD226-Fc, TIGIT-Fc, or CD96-Fc fusionproteins. (3B) HEK-293T cells were transiently transfected with a cDNAexpression vector encoding PVR-CD4TM-GFP, PVRL1-CD4TM-GFP,PVRL2-CD4TM-GFP, PVRL3-CD4TM-GFP, or PVRL4-CD4TM-GFP and thensubsequently mixed with soluble PVR-Fc, PVRL1-Fc, PVRL2-Fc, PVRL3-Fc, orPVRL4-Fc fusion proteins. Specific binding is indicated by the presenceof signal within the dark circle overlay on each FACS plot. (3C) Aschematic representation of the observed binding interactions betweenthe different members of the PVR family.

FIGS. 4A and 4B. Sequence of the N-terminal IgV domain of human PVR (SEQID NO:17) and human PVRL2 (SEQ ID NO:23) showing specific amino acidresidues (in bold) selected for potential alteration in a library of PVRvariants (4A) and PVRL2 variants (4B).

FIG. 5. FACS analysis of binding interactions between PVR variants andTIGIT, CD96, and CD226. HEK-293T cells were transiently transfected witha cDNA expression vector encoding PVR-CD4TM-GFP, PVR S72Nvariant-CD4TM-GFP, or PVR Q82K variant-CD4TM-GFP and then subsequentlymixed with soluble TIGIT-Fc, CD96-Fc, CD226-Fc fusion proteins, acombination of TIGIT-Fc and CD226-Fc fusion proteins, or a combinationof CD96-Fc and CD226-Fc fusion proteins.

FIG. 6. Natural Killer Cell Cytotoxicity assay. Human NK cells werepre-treated with 30 μg/ml of PVR-Fc variant Q82K (gray bar), PVR-Fcwild-type control (black bar), or medium only (white bar). Target cells(HEK-293T cells or K562 cells) were labeled with 10 μM calcein AM mixedwith the NK cells at an effector:target ratio of 12:1. Supernatants wereharvested and calcein release was quantified on a fluorometer at anexcitation of 485 nm and an emission of 535 nm.

FIG. 7. Natural Killer Cytotoxicity assay. HEK-293T or A549 cells wereseeded into plates and grown to confluence overnight. NK cells werepre-treated with 30 μg/ml of PVR-Fc variant Q82K (gray bar), PVR-Fcwild-type control (black bar), or medium only (white bar) and added tothe target cells with or without human IL-2. Culture supernatants wereharvested after 24 hours and analyzed for IFN-gamma content by ELISA(R&D Systems, Minneapolis, Minn.).

FIGS. 8A and 8B. FACS analysis of binding interactions between PVRvariants and TIGIT, CD226, and PVRL3. HEK-293T cells were transientlytransfected with a cDNA expression vector encoding (8A) PVR-CD4TM-GFP,(8A) PVR S72N variant-CD4TM-GFP, (8B) PVR Q82K variant-CD4TM-GFP, or(8B) PVR Q82K+S72N double variant-CD4TM-GFP. After 24 hours, cells weremixed with soluble TIGIT-Fc, CD226-Fc, or PVRL3-Fc fusion proteins andthen subsequently stained with PE-conjugated anti-human Fc secondaryantibody. Fusion protein binding was then analyzed by flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel agents, including, but not limitedto, polypeptides, soluble receptors, and antibodies that modulate theimmune response. The agents include agonists and antagonists ofreceptors that are members of the immunoglobulin superfamily involved incell interactions and immune response signaling. Related polypeptidesand polynucleotides, compositions comprising the agents, and methods ofmaking the agents are also provided. Methods of screening for agentsthat modulate the immune response are provided. Methods of using thenovel agents, such as methods of activating an immune response, methodsof stimulating an immune response, methods of promoting an immuneresponse, methods of increasing an immune response, methods ofactivating natural killer (NK) cells and/or T-cells, methods ofincreasing the activity of NK cells and/or T-cells, methods of promotingthe activity of NK cells and/or T-cells, methods of inhibiting tumorgrowth, and/or methods of treating cancer are further provided.

I. Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The terms “agonist” and “agonistic” as used herein refer to or describean agent that is capable of, directly or indirectly, substantiallyinducing, activating, promoting, increasing, or enhancing the biologicalactivity of a target and/or a pathway. The term “agonist” is used hereinto include any agent that partially or fully induces, activates,promotes, increases, or enhances the activity of a protein. Suitableagonists specifically include, but are not limited to, agonistantibodies or fragments thereof, soluble receptors, other fusionproteins, polypeptides, and small molecules.

The terms “antagonist” and “antagonistic” as used herein refer to ordescribe an agent that is capable of, directly or indirectly, partiallyor fully blocking, inhibiting, reducing, or neutralizing a biologicalactivity of a target and/or pathway. The term “antagonist” is usedherein to include any agent that partially or fully blocks, inhibits,reduces, or neutralizes the activity of a protein. Suitable antagonistagents specifically include, but are not limited to, antagonistantibodies or fragments thereof, soluble receptors, other fusionproteins, polypeptides, and small molecules.

The terms “modulation” and “modulate” as used herein refer to a changeor an alteration in a biological activity. Modulation includes, but isnot limited to, stimulating or inhibiting an activity. Modulation may bean increase or a decrease in activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties associated with the activity of a protein, apathway, a system, or other biological targets of interest.

As used herein, the term “soluble receptor” refers to an extracellularfragment of a receptor protein preceding the first transmembrane domainof the receptor that can be secreted from a cell in soluble form. Theterm “soluble receptor” encompasses a molecule comprising the entireextracellular domain, or a fragment of the extracellular domain.

As used herein, the term “linker” or “linker region” refers to a linkerinserted between a first polypeptide (e.g., a PVR component) and asecond polypeptide (e.g., a Fc region). In some embodiments, the linkeris a peptide linker. Linkers should not adversely affect the expression,secretion, or bioactivity of the polypeptides. Preferably, linkers arenot antigenic and do not elicit an immune response.

The terms “selectively binds” or “specifically binds” mean that abinding agent reacts or associates more frequently, more rapidly, withgreater duration, with greater affinity, or with some combination of theabove to the epitope, protein, or target molecule than with alternativesubstances, including related and unrelated proteins. In certainembodiments “specifically binds” means, for instance, that a bindingagent binds a protein or target with a K_(D) of about 0.1 mM or less,but more usually less than about 1 μM. In certain embodiments,“specifically binds” means that a binding agent binds a target with aK_(D) of at least about 0.1 μM or less, at least about 0.01 μM or less,or at least about 1 nM or less. Because of the sequence identity betweenhomologous proteins in different species, specific binding can include abinding agent that recognizes a protein or target in more than onespecies. Likewise, because of homology within certain regions ofpolypeptide sequences of different proteins, specific binding caninclude a binding agent that recognizes more than one protein or target.It is understood that, in certain embodiments, a binding agent thatspecifically binds a first target may or may not specifically bind asecond target. As such, “specific binding” does not necessarily require(although it can include) exclusive binding, i.e. binding to a singletarget. Thus, a binding agent may, in certain embodiments, specificallybind more than one target. In certain embodiments, multiple targets maybe bound by the same antigen-binding site on the binding agent. Forexample, an antibody may, in certain instances, comprise two identicalantigen-binding sites, each of which specifically binds the same epitopeon two or more proteins. In certain alternative embodiments, an antibodymay be bispecific and comprise at least two antigen-binding sites withdiffering specificities. By way of non-limiting example, a bispecificantibody may comprise one antigen-binding site that recognizes anepitope on one protein and further comprise a second, differentantigen-binding site that recognizes a different epitope on a secondprotein. Generally, but not necessarily, reference to binding meansspecific binding.

The terms “polypeptide” and “peptide” and “protein” are usedinterchangeably herein and refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids), as well as other modifications known in the art. It isunderstood that, because the polypeptides of this invention may be basedupon antibodies or other members of the immunoglobulin superfamily, incertain embodiments, the polypeptides can occur as single chains or asassociated chains.

The terms “polynucleotide” and “nucleic acid” and “nucleic acidmolecule” are used interchangeably herein and refer to polymers ofnucleotides of any length, and include DNA and RNA. The nucleotides canbe deoxyribonucleotides, ribonucleotides, modified nucleotides or bases,and/or their analogs, or any substrate that can be incorporated into apolymer by DNA or RNA polymerase.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software that may be used to obtain alignments of aminoacid or nucleotide sequences are well-known in the art. These include,but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG WisconsinPackage, and variants thereof. In some embodiments, two nucleic acids orpolypeptides of the invention are substantially identical, meaning theyhave at least 70%, at least 75%, at least 80%, at least 85%, at least90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotideor amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using a sequence comparison algorithm or byvisual inspection. In some embodiments, identity exists over a region ofthe sequences that is at least about 10, at least about 20, at leastabout 40-60 residues, at least about 60-80 residues in length or anyintegral value there between. In some embodiments, identity exists overa longer region than 60-80 residues, such as at least about 80-100residues, and in some embodiments the sequences are substantiallyidentical over the full length of the sequences being compared, such asthe coding region of a nucleotide sequence.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Generally,conservative substitutions in the sequences of the polypeptides, solublereceptors, and/or antibodies of the invention do not abrogate thebinding of the polypeptide, soluble receptor, or antibody containing theamino acid sequence, to the target binding site. Methods of identifyingnucleotide and amino acid conservative substitutions which do noteliminate binding are well-known in the art.

The term “vector” as used herein means a construct, which is capable ofdelivering, and usually expressing, one or more gene(s) or sequence(s)of interest in a host cell. Examples of vectors include, but are notlimited to, viral vectors, naked DNA or RNA expression vectors, plasmid,cosmid, or phage vectors, DNA or RNA expression vectors associated withcationic condensing agents, and DNA or RNA expression vectorsencapsulated in liposomes.

A polypeptide, soluble receptor, antibody, polynucleotide, vector, cell,or composition which is “isolated” is a polypeptide, soluble receptor,antibody, polynucleotide, vector, cell, or composition which is in aform not found in nature. Isolated polypeptides, soluble receptors,antibodies, polynucleotides, vectors, cells, or compositions includethose which have been purified to a degree that they are no longer in aform in which they are found in nature. In some embodiments, apolypeptide, soluble receptor, antibody, polynucleotide, vector, cell,or composition which is isolated is substantially pure.

The term “substantially pure” as used herein refers to material which isat least 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The term “immune response” as used herein includes responses from boththe innate immune system and the adaptive immune system. It includesboth T-cell and B-cell responses (e.g., cell-mediated and/or humoralimmune responses), as well as responses from other cells of the immunesystem such as natural killer (NK) cells, monocytes, macrophages, etc.

The terms “cancer” and “cancerous” as used herein refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, blastoma, sarcoma, andhematologic cancers such as lymphoma and leukemia.

The terms “tumor” and “neoplasm” as used herein refer to any mass oftissue that results from excessive cell growth or proliferation, eitherbenign (noncancerous) or malignant (cancerous) including pre-cancerouslesions.

The term “metastasis” as used herein refers to the process by which acancer spreads or transfers from the site of origin to other regions ofthe body with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “tumorinitiating cell” are used interchangeably herein and refer to cells froma cancer or tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more types ofdifferentiated cell progeny wherein the differentiated cells havereduced proliferative or developmental potential; and (3) are capable ofsymmetric cell divisions for self-renewal or self-maintenance. Theseproperties confer on the cancer stem cells the ability to form orestablish a tumor or cancer upon serial transplantation into anappropriate host (e.g., a mouse) compared to the majority of tumor cellsthat fail to form tumors. Cancer stem cells undergo self-renewal versusdifferentiation in a chaotic manner to form tumors with abnormal celltypes that can change over time as mutations occur.

The terms “cancer cell” and “tumor cell” refer to the total populationof cells derived from a cancer or tumor or pre-cancerous lesion,including both non-tumorigenic cells, which comprise the bulk of thecancer cell population, and tumorigenic stem cells (cancer stem cells).As used herein, the terms “cancer cell” or “tumor cell” will be modifiedby the term “non-tumorigenic” when referring solely to those cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “tumorigenic” as used herein refers to the functional featuresof a cancer stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells).

The term “tumorigenicity” as used herein refers to the ability of arandom sample of cells from the tumor to form palpable tumors uponserial transplantation into appropriate hosts (e.g., mice).

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to, humans, non-human primates, canines, felines, rodents,and the like, which is to be the recipient of a particular treatment.Typically, the terms “subject” and “patient” are used interchangeablyherein in reference to a human subject.

The term “pharmaceutically acceptable” refers to a substance approved orapprovable by a regulatory agency of the Federal or a state governmentor listed in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, including humans.

The terms “pharmaceutically acceptable excipient, carrier or adjuvant”or “acceptable pharmaceutical carrier” refer to an excipient, carrier oradjuvant that can be administered to a subject, together with at leastone binding agent (e.g., an antibody) of the present disclosure, andwhich does not destroy the pharmacological activity thereof and isnontoxic when administered in doses sufficient to deliver a therapeuticeffect.

The terms “effective amount” or “therapeutically effective amount” or“therapeutic effect” refer to an amount of a binding agent, a solublereceptor, an antibody, polypeptide, polynucleotide, small organicmolecule, or other drug effective to “treat” a disease or disorder in asubject such as, a mammal. In the case of cancer or a tumor, thetherapeutically effective amount of an agent (e.g., soluble receptor orantibody) has a therapeutic effect and as such can boost the immuneresponse, boost the anti-tumor response, increase cytolytic activity ofimmune cells, increase killing of tumor cells by immune cells, reducethe number of tumor cells; decrease tumorigenicity, tumorigenicfrequency or tumorigenic capacity; reduce the number or frequency ofcancer stem cells; reduce the tumor size; reduce the cancer cellpopulation; inhibit or stop cancer cell infiltration into peripheralorgans including, for example, the spread of cancer into soft tissue andbone; inhibit and stop tumor or cancer cell metastasis; inhibit and stoptumor or cancer cell growth; relieve to some extent one or more of thesymptoms associated with the cancer; reduce morbidity and mortality;improve quality of life; or a combination of such effects.

The terms “treating” or “treatment” or “to treat” or “alleviating” or“to alleviate” refer to both (1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and (2) prophylactic or preventativemeasures that prevent or slow the development of a targeted pathologiccondition or disorder. Thus those in need of treatment include thosealready with the disorder; those prone to have the disorder; and thosein whom the disorder is to be prevented. In the case of cancer or atumor, a subject is successfully “treated” according to the methods ofthe present invention if the patient shows one or more of the following:an increased immune response, an increased anti-tumor response,increased cytolytic activity of immune cells, increased killing of tumorcells by immune cells, a reduction in the number of or complete absenceof cancer cells; a reduction in the tumor size; inhibition of or anabsence of cancer cell infiltration into peripheral organs including thespread of cancer cells into soft tissue and bone; inhibition of or anabsence of tumor or cancer cell metastasis; inhibition or an absence ofcancer growth; relief of one or more symptoms associated with thespecific cancer; reduced morbidity and mortality; improvement in qualityof life; reduction in tumorigenicity; reduction in the number orfrequency of cancer stem cells; or some combination of effects.

As used in the present disclosure and claims, the singular forms “a”,“an” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting of” and/or “consisting essentially of” are also provided.It is also understood that wherever embodiments are described hereinwith the language “consisting essentially of” otherwise analogousembodiments described in terms of “consisting of” are also provided.

As used herein, reference to “about” or “approximately” a value orparameter includes (and describes) embodiments that are directed to thatvalue or parameter. For example, description referring to “about X”includes description of “X”.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

II. Binding Agents

The present invention provides agents that bind members of theimmunoglobulin superfamily, particularly the PVR family. The PVR familyincludes, but is not limited to, poliovirus receptor (PVR), poliovirusreceptor-related protein 1 (PVRL1), poliovirus receptor-related protein2 (PVRL2), poliovirus receptor-related protein 3 (PVRL3), poliovirusreceptor-related protein 4 (PVRL4), T cell immunoreceptor with Ig andITIM domains (TIGIT), CD226, and CD96. These proteins are all generallyrelated in both structure and function. The receptors are type Itransmembrane proteins, which typically consist of an extracellulardomain (ECD) containing one or more immunoglobulin (Ig)-like domains, asingle transmembrane domain, and a cytoplasmic tail. The receptorsmediate interactions through their N-terminal Ig-like domains, whichcommonly bind other Ig-like domains on an opposing cell surface(homophilic interaction), and also interact with integrins andcarbohydrates (heterophilic interaction) (Wong et al., 2012, Int. J.Cell Biol.; epub).

Human poliovirus receptor (PVR) is a 70 kD protein that contains threeextracellular Ig-like domains, a transmembrane domain, and a cytoplasmictail. The Ig-like domains include an N-terminal V-type domain followedby two C2-type domains. PVR is primarily found on endothelial cells,monocytes, epithelial cells, and central nervous system cells. PVR ininvolved in cell-cell and cell-matrix interactions with CD226, CD96,PVRL3, and vitronectin. PVR is also known as CD155, nectin-like 5, andNECL-5.

Human poliovirus receptor-related proteins 1-4 (PVRL1-4) all have astructure similar to PVR, i.e., three Ig-like domains including anN-terminal V-type domain followed by two C2-type domains, atransmembrane domain, and a cytoplasmic tail. PVRL1 is broadly expressedon endothelial cells, epithelial cells, neuronal cells, megakaryoctyes,and CD34-positive stem cells. PVRL1 functions as a receptor for herpessimplex viruses (HSV-1 and HSV-2) and is involved in the formation ofcell junctions. PVRL1 is also known as CD111, nectin-1, HVEC, HLGR, andPRR1. Similar to PVRL1, PVRL2 is broadly expressed on endothelial cells,epithelial cells, neuronal cells, megakaryoctyes, and CD34-positive stemcells and functions as a receptor for HSV. In addition, it is involvedin the formation of cell junctions and interacts with CD226 and otherPVR family members. PVRL2 is also known as CD112, nectin-2, I-WEB andPRR2. PVRL3 and PVRL4 appear to be only weakly expressed on most normalcells, however, similar to PVRL1 and PVRL2, PVRL3 and PVRL4 are involvedin the formation of cell junctions. In addition, PVRL4 has beenidentified as a receptor of the measles virus. PVRL3 is also known asCD113 and nectin-3, while PVRL4 is also known as nectin-4, LNIR, andPRR4.

CD226 is a ˜65 kD glycoprotein that contains two Ig-like domainsincluding two C2-type domains, followed by a transmembrane domain, and acytoplasmic tail containing an immunoreceptor tyrosine-based activationmotif (ITAM). CD226 has been observed on the surface of natural killer(NK) cells, monocytes, macrophages, T-cells, megakaryocytes, and asubset of B-cells. CD226 binds PVR and PVRL2, and appears to be involvedin activation of NK cells and T-cells. This receptor is also known asDNAM-1, PTA-1, and TLiSA1.

TIGIT is a 26 kD protein that contains one Ig-like V-type domain,followed by a transmembrane domain, and a cytoplasmic tail containingtwo immunoreceptor tyrosine-based inhibition motifs (ITIM). TIGIT hasbeen observed on the surface of NK cells and most activated T-cells, butis low or negative on naive lymphocytes. TIGIT binds PVR, PVRL2, PVRL3,and PVRL4, and appears to have an inhibitory function on both T-cellsand NK cells. This receptor is also known as VSIG9, Vstm3, and WUCAM.

CD96 is a 160 kD protein that contains three Ig-like domains includingtwo V-type domains and one C2-type domain, followed by a transmembranedomain, and a cytoplasmic tail containing an ITIM motif. CD96 has beenshown to be expressed on the surface of NK cells and T-cells. CD96 bindsto PVR and it is believed that the predominant function of CD96 is tomediate adhesion of NK cells to other cells expressing PVR. However, thepresence of an ITIM suggests that CD96 may also have an inhibitoryfunction. This receptor is also known as tactile.

The full-length amino acid (aa) sequences of human PVR, PVRL1-4, TIGIT,CD226, and CD96 are known in the art and are provided herein as SEQ IDNO:1 (PVR), SEQ ID NO:2 (PVRL1), SEQ ID NO:3 (PVRL2), SEQ ID NO:4(PVRL3), SEQ ID NO:5 (PVRL4), SEQ ID NO:6 (TIGIT), SEQ ID NO:7 (CD96),and SEQ ID NO:8 (CD226). As used herein, reference to amino acidpositions corresponding to a “wild-type protein” refer to the numberingof full-length amino acid sequences including the signal sequence.

In certain embodiments, the binding agent is a polypeptide. In someembodiments, the binding agent comprises a soluble receptor. In certainembodiments, the binding agent is a soluble receptor. In certainembodiments, the binding agent is a bispecific agent. In certainembodiments, the binding agent (e.g., a soluble receptor or apolypeptide) comprises a PVR variant. As used herein, a “variant”protein comprises substitutions, deletions, and/or additions in one ormore amino acids corresponding to amino acids of the wild-type protein.In some embodiments, the PVR variant comprises one or more Ig-likedomains of human PVR. In certain embodiments, the PVR variant comprisesan N-terminal IgV domain of human PVR, wherein the PVR variant comprisesone or more amino acid substitutions as compared to wild-type PVR. Incertain embodiments, the PVR variant consists essentially of anN-terminal IgV domain of human PVR, wherein the PVR variant comprisesone or more amino acid substitutions as compared to wild-type PVR. Insome embodiments, the PVR variant comprises an N-terminal IgV domain andone IgC2 domain of human PVR, wherein the PVR variant comprises one ormore amino acid substitutions as compared to wild-type PVR. In someembodiments, the PVR variant comprises an N-terminal IgV domain and bothIgC2 domains of human PVR, wherein the PVR variant comprises one or moreamino acid substitutions as compared to wild-type PVR. In someembodiments, the PVR variant comprises substitutions in one or moreamino acids corresponding to amino acids 40-143 of wild-type PVR. Insome embodiments, the PVR variant comprises substitutions in one or moreamino acids corresponding to amino acids 60-90 of wild-type PVR. In someembodiments, the PVR variant comprises substitutions in one or moreamino acids corresponding to amino acids 125-133 of wild-type PVR. Insome embodiments, the PVR variant comprises substitutions in one or moreamino acids corresponding to amino acids 60-90 and 125-133 of wild-typePVR. In some embodiments, the PVR variant comprises substitutions in oneor more amino acids corresponding to amino acids 65, 67, 72, 73, 74, 81,82, 84, and 85 of wild-type PVR. In some embodiments, the PVR variantcomprises a substitution in an amino acid corresponding to amino acid 72of wild-type PVR. In some embodiments, the PVR variant comprises asubstitution in an amino acid corresponding to amino acid 82 ofwild-type PVR. In some embodiments, the PVR variant comprisessubstitutions in one or more amino acids corresponding to amino acids 72and 82 of wild-type PVR. In some embodiments, the PVR variant comprisesan amino acid sequence selected from the group consisting of: SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

In certain embodiments, the binding agent (e.g., a soluble receptor or apolypeptide) comprises a PVRL1 variant. In some embodiments, the PVRL1variant comprises one or more Ig-like domains of human PVRL1. In certainembodiments, the PVRL1 variant comprises an N-terminal IgV domain ofhuman PVRL1, wherein the PVRL1 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL1. In certain embodiments,the PVRL1 variant consists essentially of an N-terminal IgV domain ofhuman PVRL1, wherein the PVRL1 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL1. In some embodiments, thePVRL1 variant comprises an N-terminal IgV domain and one IgC2 domain ofhuman PVRL1, wherein the PVRL1 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL1. In some embodiments, thePVRL1 variant comprises an N-terminal IgV domain and both IgC2 domainsof human PVRL1, wherein the PVRL1 variant comprises one or more aminoacid substitutions as compared to wild-type PVRL1. In some embodiments,the PVRL1 variant comprises substitutions in one or more amino acidscorresponding to amino acids 41-144 of wild-type PVRL1. In someembodiments, the PVRL1 variant comprises substitutions in one or moreamino acids corresponding to amino acids 61-93 of wild-type PVRL1. Insome embodiments, the PVRL1 variant comprises substitutions in one ormore amino acids corresponding to amino acids 126-134 of wild-typePVRL1. In some embodiments, the PVRL1 variant comprises substitutions inone or more amino acids corresponding to amino acids 61-93 and 126-134of wild-type PVRL1.

In certain embodiments, the binding agent (e.g., a soluble receptor or apolypeptide) comprises a PVRL2 variant. In some embodiments, the PVRL2variant comprises one or more Ig-like domains of human PVRL2. In certainembodiments, the PVRL2 variant comprises an N-terminal IgV domain ofhuman PVRL2, wherein the PVRL2 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL2. In certain embodiments,the PVRL2 variant consists essentially of an N-terminal IgV domain ofhuman PVRL2, wherein the PVRL2 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL2. In some embodiments, thePVRL2 variant comprises an N-terminal IgV domain and one IgC2 domain ofhuman PVRL2, wherein the PVRL2 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL2. In some embodiments, thePVRL2 variant comprises an N-terminal IgV domain and both IgC2 domainsof human PVRL2, wherein the PVRL2 variant comprises one or more aminoacid substitutions as compared to wild-type PVRL2. In some embodiments,the PVRL2 variant comprises substitutions in one or more amino acidscorresponding to amino acids 45-160 of wild-type PVRL2. In someembodiments, the PVRL2 variant comprises substitutions in one or moreamino acids corresponding to amino acids 64-97 of wild-type PVRL2. Insome embodiments, the PVRL2 variant comprises substitutions in one ormore amino acids corresponding to amino acids 142-150 of wild-typePVRL2. In some embodiments, the PVRL2 variant comprises substitutions inone or more amino acids corresponding to amino acids 64-97 and 142-150of wild-type PVRL2.

In certain embodiments, the binding agent (e.g., a soluble receptor or apolypeptide) comprises a PVRL3 variant. In some embodiments, the PVRL3variant comprises one or more Ig-like domains of human PVRL3. In certainembodiments, the PVRL3 variant comprises an N-terminal IgV domain ofhuman PVRL3, wherein the PVRL3 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL3. In certain embodiments,the PVRL3 variant consists essentially of an N-terminal IgV domain ofhuman PVRL3, wherein the PVRL3 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL3. In some embodiments, thePVRL3 variant comprises an N-terminal IgV domain and one IgC2 domain ofhuman PVRL3, wherein the PVRL3 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL3. In some embodiments, thePVRL3 variant comprises an N-terminal IgV domain and both IgC2 domainsof human PVRL3, wherein the PVRL3 variant comprises one or more aminoacid substitutions as compared to wild-type PVRL3. In some embodiments,the PVRL3 variant comprises substitutions in one or more amino acidscorresponding to amino acids 68-168 of wild-type PVRL3. In someembodiments, the PVRL3 variant comprises substitutions in one or moreamino acids corresponding to amino acids 86-117 of wild-type PVRL3. Insome embodiments, the PVRL3 variant comprises substitutions in one ormore amino acids corresponding to amino acids 150-158 of wild-typePVRL3. In some embodiments, the PVRL3 variant comprises substitutions inone or more amino acids corresponding to amino acids 86-117 and 150-158of wild-type PVRL3.

In certain embodiments, the binding agent (e.g., a soluble receptor or apolypeptide) comprises a PVRL4 variant. In some embodiments, the PVRL4variant comprises one or more Ig-like domains of human PVRL4. In certainembodiments, the PVRL4 variant comprises an N-terminal IgV domain ofhuman PVRL4, wherein the PVRL4 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL4. In certain embodiments,the PVRL4 variant consists essentially of an N-terminal IgV domain ofhuman PVRL4, wherein the PVRL4 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL4. In some embodiments, thePVRL4 variant comprises an N-terminal IgV domain and one IgC2 domain ofhuman PVRL4, wherein the PVRL4 variant comprises one or more amino acidsubstitutions as compared to wild-type PVRL4. In some embodiments, thePVRL4 variant comprises an N-terminal IgV domain and both IgC2 domainsof human PVRL4, wherein the PVRL4 variant comprises one or more aminoacid substitutions as compared to wild-type PVRL4. In some embodiments,the PVRL4 variant comprises substitutions in one or more amino acidscorresponding to amino acids 42-147 of wild-type PVRL4. In someembodiments, the PVRL4 variant comprises substitutions in one or moreamino acids corresponding to amino acids 62-94 of wild-type PVRL4. Insome embodiments, the PVRL4 variant comprises substitutions in one ormore amino acids corresponding to amino acids 129-137 of wild-typePVRL4. In some embodiments, the PVRL4 variant comprises substitutions inone or more amino acids corresponding to amino acids 62-94 and 129-137of wild-type PVRL4.

The extracellular domains (ECD) for PVR, PVRL1, PVRL2, PVRL3, PVRL4,TIGIT, CD96, and CD226 are provided as SEQ ID NOs:9-16 (withoutpredicted signal sequences). Those of skill in the art may differ intheir understanding of the exact amino acids corresponding to thevarious ECD domains. Thus, the N-terminus and/or C-terminus of the ECDsdescribed herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more amino acids. This is also true for the individual Ig-typedomains within the ECDs.

Human TIGIT and human CD96 are inhibitory receptors which mediate theiractivity via their ITIMs and are believed to have the ability to inhibitimmune responses. In contrast, human CD226 is an activation receptorwhich mediates its activity via an ITAM and is believed to have theability to activate immune responses. TIGIT, CD96 and CD226 are allexpressed on NK cells and T-cells. All three receptors have been shownto bind PVR, with TIGIT having the highest affinity for PVR as comparedto CD96 and CD226. In many situations, it appears that the inhibitoryeffects of TIGIT are dominant and an immune response to antigenicstimulation (e.g., a tumor, a virus, an infection) is reduced orsuppressed. Without being bound by theory, it is proposed that throughthe manipulation of the inhibitory receptors TIGIT and/or CD96, that astrong immune response could be activated and/or increased. For example,a strong immune response could be achieved using binding agents thatspecifically interact with TIGIT, but do not activate signaling (i.e.,“blocking agents”), wherein the agents do not bind and/or affect theactivation of CD226, allowing for an increase in the activity of, forexample, NK cells and/or T-cells. The immune response could bestrengthened if the binding agents specifically interact with both TIGITand CD96, without activating any inhibitory signaling from thesemolecules. This would allow CD226 signaling to be dominant, resulting ina strong or stronger immune response.

Thus, in some embodiments, the binding agent (e.g., a soluble receptor)interferes with the interaction between PVR and TIGIT. In someembodiments, the binding agent interferes with the interaction betweenPVR and TIGIT and the interaction between PVR and CD96. In someembodiments, the binding agent interferes with the interaction betweenPVR and CD96. In some embodiments, the binding agent interferes with theinteraction between PVR and TIGIT, but does not interfere with theinteraction between PVR and CD226. In some embodiments, the bindingagent interferes with the interaction between PVR and TIGIT and theinteraction between PVR and CD96, but does not interfere with theinteraction between PVR and CD226. In some embodiments, the bindingagent interferes with the interaction between PVR and CD96, but does notinterfere with the interaction between PVR and CD226. In someembodiments, the binding agent comprises a soluble receptor comprising aPVR variant, wherein the binding agent interferes with the interactionbetween PVR and TIGIT, the interaction between PVR and CD96, and doesnot interfere with the interaction between PVR and CD226. In someembodiments, the binding agent comprises a soluble receptor comprising aPVRL2 variant, wherein the binding agent interferes with the interactionbetween PVRL2 and TIGIT and does not interfere with the interactionbetween PVRL2 and CD226.

In some embodiments, the binding agent (e.g., a soluble receptor)specifically binds the extracellular domain of human TIGIT. In someembodiments, the binding agent specifically binds the extracellulardomain of human CD96. In some embodiments, the binding agentspecifically binds the extracellular domain of human TIGIT and binds theextracellular domain of CD96. In some embodiments, the binding agentspecifically binds the extracellular domain of human TIGIT and does notbind (or binds weakly to) the extracellular domain of human CD226. Insome embodiments, the binding agent specifically binds the extracellulardomain of human CD96 and does not bind (or binds weakly to) theextracellular domain of CD226. In some embodiments, the binding agentspecifically binds the extracellular domain of human TIGIT and binds theextracellular domain of CD96, and does not bind (or binds weakly to) theextracellular domain of human CD226. In some embodiments, the bindingagent comprises a soluble receptor comprising a PVR variant, wherein thebinding agent specifically binds TIGIT and CD96, and does not bind (orbinds weakly to) CD226. In some embodiments, the binding agent comprisesa soluble receptor comprising a PVRL2 variant, wherein the binding agentspecifically binds TIGIT and does not bind (or binds weakly to) CD226.

In some embodiments, the binding agent (e.g., a soluble receptor)specifically binds the extracellular domain of human TIGIT and inhibitsor interferes with the interaction (e.g., binding) between PVR andTIGIT. In some embodiments, the binding agent specifically binds theextracellular domain of human TIGIT and the extracellular domain ofhuman CD96 and inhibits or interferes with the interaction (e.g.,binding) between PVR and TIGIT and the interaction (e.g., binding)between PVR and CD96. In some embodiments, the binding agentspecifically binds the extracellular domain of human TIGIT and inhibitsor interferes with the interaction (e.g., binding) between PVR andTIGIT, but does not bind (or binds weakly to) the extracellular domainof human CD226 and does not inhibit or interfere with the interaction(e.g., binding) between PVR and CD226. In some embodiments, the bindingagent specifically binds the extracellular domain of human TIGIT and theextracellular domain of human CD96 and inhibits or interferes with theinteraction (e.g., binding) between PVR and TIGIT and the interaction(e.g., binding) between PVR and CD96, but does not bind (or binds weaklyto) the extracellular domain of human CD226 and does not inhibit orinterfere with the interaction (e.g., binding) between PVR and CD226. Insome embodiments, the binding agent comprises a soluble receptorcomprising a PVR variant, wherein the soluble receptor comprising a PVRvariant specifically binds the extracellular domain of human TIGIT andthe extracellular domain of human CD96 and inhibits or interferes withthe interaction (e.g., binding) between PVR and TIGIT and theinteraction (e.g., binding) between PVR and CD96, but does not bind (orbinds weakly to) the extracellular domain of human CD226 and does notinhibit or interfere with the interaction (e.g., binding) between PVRand CD226. In some embodiments, the binding agent comprises a solublereceptor comprising a PVRL2 variant, wherein the soluble receptorcomprising a PVRL2 variant specifically binds the extracellular domainof human TIGIT and inhibits or interferes with the interaction (e.g.,binding) between PVRL2 and TIGIT and the interaction (e.g., binding)between PVR and TIGIT, but does not bind (or binds weakly to) theextracellular domain of human CD226 and does not inhibit or interferewith the interaction (e.g., binding) between PVR and CD226.

In some embodiments, the binding agent (e.g., a soluble receptor)comprises a PVR variant that specifically binds the extracellular domainof human TIGIT, but does not bind (or binds weakly to) the extracellulardomain of human CD226. In some embodiments, the binding agent (e.g., asoluble receptor) comprises a PVR variant that specifically binds theextracellular domain of human TIGIT and specifically binds theextracellular domain of human CD96, but does not bind (or binds weaklyto) the extracellular domain of human CD226. In some embodiments, thePVR variant is a PVR variant described herein. In some embodiments, thePVR variant comprises a sequence selected from the group consisting ofSEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

In some embodiments, the binding agent (e.g., a soluble receptor or apolypeptide) comprises a PVRL2 variant that specifically binds theextracellular domain of human TIGIT, but does not bind (or binds weaklyto) the extracellular domain of human CD226. In some embodiments, thePVRL2 variant is a PVRL2 variant described herein. In some embodiments,the PVRL2 variant comprises SEQ ID NO:38.

In some embodiments, the binding agent specifically binds theextracellular domain of human TIGIT and inhibits or interferes with theinteraction (e.g., binding) between PVRL2 and TIGIT. In someembodiments, the binding agent specifically binds the extracellulardomain of human TIGIT and inhibits or interferes with the interaction(e.g., binding) between PVRL2 and TIGIT, but does not bind (or bindsweakly to) the extracellular domain of human CD226 and does not inhibitor interfere with the interaction (e.g., binding) between PVRL2 andCD226.

In some embodiments, the binding agent (e.g., a soluble receptor)comprises a PVRL3 variant that specifically binds the extracellulardomain of human TIGIT. In some embodiments, the binding agent (e.g., asoluble receptor) comprises a PVRL4 variant that specifically binds theextracellular domain of human TIGIT.

In some embodiments, the binding agent specifically binds theextracellular domain of human TIGIT and inhibits or interferes with theinteraction (e.g., binding) between PVRL3 and TIGIT. In someembodiments, the binding agent specifically binds the extracellulardomain of human TIGIT and inhibits or interferes with the interaction(e.g., binding) between PVRL4 and TIGIT.

In some embodiments, the binding agent is a TIGIT-binding agentcomprising one or more Ig-like domains of a variant human PVR. In someembodiments, the binding agent is a CD96-binding agent comprising one ormore Ig-like domains of a variant human PVR. In some embodiments, thebinding agent is a TIGIT and CD96-binding agent comprising one or moreIg-like domains of a variant human PVR. In some embodiments, theTIGIT-binding agent comprises a variant human PVR and does not bind (orbinds weakly to) CD226.

In some embodiments, the binding agent (e.g., a soluble receptor) is afusion protein. As used herein, a “fusion protein” is a hybrid proteinexpressed by a nucleic acid molecule comprising nucleotide sequences ofat least two genes. In certain embodiments, the binding agent, such as asoluble receptor or a polypeptide, further comprises a non-PVRpolypeptide. In some embodiments, soluble receptors may include a PVRfamily member ECD or fragment thereof (e.g., Ig-like domain) linked tonon-PVR polypeptides including, but not limited to, a human Fc region,protein tags (e.g., myc, FLAG, GST), other endogenous proteins orprotein fragments, or any other useful protein sequences including anylinker region between an ECD and a second polypeptide. In certainembodiments, the non-PVR polypeptide comprises a human Fc region. Incertain embodiments, the non-PVR polypeptide consists essentially of ahuman Fc region. In certain embodiments, the non-PVR polypeptideconsists of a human Fc region. The Fc region can be obtained from any ofthe classes of immunoglobulin, IgG, IgA, IgM, IgD and IgE. In someembodiments, the Fc region is a human IgG1 Fc region. In someembodiments, the Fc region is a human IgG2 Fc region. In someembodiments, the Fc region is a wild-type Fc region. In someembodiments, the Fc region is a wild-type Fc region containing naturalamino acid variations. In some embodiments, the Fc region is a mutatedor modified Fc region. In some embodiments, the Fc region is truncatedat the N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more aminoacids, (e.g., in the hinge domain). In some embodiments, the Fc regionis truncated at the C-terminal end by one or more amino acids, (e.g.,missing the C-terminal lysine). In some embodiments, an amino acid inthe hinge domain is changed to hinder undesirable disulfide bondformation. In some embodiments, a cysteine is replaced with a differentamino acid to hinder undesirable disulfide bond formation. In someembodiments, a cysteine is replaced with a serine to hinder undesirabledisulfide bond formation. In some embodiments, the Fc region is modifiedto promote formation of heteromultimers or heterodimeric molecules. Incertain embodiments, the non-PVR polypeptide comprises SEQ ID NO:26, SEQID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ IDNO:48. In certain embodiments, the non-PVR polypeptide consistsessentially of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45,SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48.

In certain embodiments, the binding agent (e.g., a soluble receptor) isa fusion protein comprising at least a fragment of a PVR variant ECD (orPVRL1-4 variant ECDs) and a Fc region. In some embodiments, theC-terminus of the PVR variant ECD (or fragment thereof) is linked to theN-terminus of the immunoglobulin Fc region. In some embodiments, the PVRvariant ECD (or fragment thereof) is directly linked to the Fc region(i.e. without an intervening peptide linker). In some embodiments, thePVR variant ECD (or fragment thereof) is linked to the Fc region via apeptide linker.

As used herein, the term “linker” refers to a linker inserted between afirst polypeptide (e.g., a PVR variant ECD or a fragment thereof) and asecond polypeptide (e.g., a Fc region). In some embodiments, the linkeris a peptide linker. Linkers should not adversely affect the expression,secretion, or bioactivity of the fusion protein. Linkers should not beantigenic and should not elicit an immune response. Suitable linkers areknown to those of skill in the art and often include mixtures of glycineand serine residues and often include amino acids that are stericallyunhindered. Other amino acids that can be incorporated into usefullinkers include threonine and alanine residues. Linkers can range inlength, for example from 1-50 amino acids in length, 1-22 amino acids inlength, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3amino acids in length. Linkers may include, but are not limited to,SerGly, GGSG, GSGS, GGGS, S(GGS)n where n is 1-7, GRA, poly(Gly),poly(Ala), ESGGGGVT (SEQ ID NO:33), LESGGGGVT (SEQ ID NO:34), GRAQVT(SEQ ID NO:35), WRAQVT (SEQ ID NO:36), and ARGRAQVT (SEQ ID NO:37). Asused herein, a linker is an intervening peptide sequence that does notinclude amino acid residues from either the C-terminus of the firstpolypeptide (e.g., a PVR variant ECD or portion thereof) or theN-terminus of the second polypeptide (e.g., a Fc region).

In some embodiments, the binding agent is a fusion protein comprising afirst polypeptide comprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, or SEQ ID NO:38, and a second polypeptide comprising SEQID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, or SEQ ID NO:48. In some embodiments, the binding agent is afusion protein comprising a first polypeptide comprising SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:38, and a secondpolypeptide comprising SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. Insome embodiments, the binding agent is a fusion protein comprising afirst polypeptide comprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, or SEQ ID NO:38, and a second polypeptide comprising SEQID NO:29, SEQ ID NO:43, or SEQ ID NO:44. In some embodiments, thebinding agent is a fusion protein comprising a first polypeptidecomprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, orSEQ ID NO:38, and a second polypeptide comprising SEQ ID NO:30. In someembodiments, the binding agent is a fusion protein comprising a firstpolypeptide comprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, or SEQ ID NO:38, and a second polypeptide comprising SEQ IDNO:31. In some embodiments, the binding agent is a fusion proteincomprising a first polypeptide comprising SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:38, and a second polypeptidecomprising SEQ ID NO:45 or SEQ ID NO:46. In some embodiments, thebinding agent is a fusion protein comprising a first polypeptidecomprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, orSEQ ID NO:38, and a second polypeptide comprising SEQ ID NO:47 or SEQ IDNO:48. In some embodiments, the binding agent is a fusion proteincomprising a first polypeptide comprising SEQ ID NO:18 and a secondpolypeptide comprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48. In some embodiments,the binding agent is a fusion protein comprising a first polypeptidecomprising SEQ ID NO:19 and a second polypeptide comprising SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, or SEQ ID NO:48. In some embodiments, the binding agent is afusion protein comprising a first polypeptide comprising SEQ ID NO:20and a second polypeptide comprising SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:43, SEQ IDNO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48. Insome embodiments, the binding agent is a fusion protein comprising afirst polypeptide comprising SEQ ID NO:21 and a second polypeptidecomprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQID NO:30, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, or SEQ ID NO:48.

In some embodiments, the binding agent comprises a first polypeptidecomprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, orSEQ ID NO:38, and a second polypeptide comprising SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ IDNO:48, wherein the first polypeptide is directly linked to the secondpolypeptide. In some embodiments, the binding agent comprises a firstpolypeptide comprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQID NO:21, and a second polypeptide comprising SEQ ID NO:30 or SEQ IDNO:31, wherein the first polypeptide is directly linked to the secondpolypeptide.

In some embodiments, the binding agent comprises a first polypeptidecomprising SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, orSEQ ID NO:38, and a second polypeptide comprising SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ IDNO:48, wherein the first polypeptide is connected to the secondpolypeptide by a linker. In some embodiments, the binding agentcomprises a first polypeptide comprising SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20, or SEQ ID NO:21, and a second polypeptide comprising SEQ IDNO:30 or SEQ ID NO:31, wherein the first polypeptide is connected to thesecond polypeptide by a linker.

In some embodiments, the binding agent comprises a first polypeptidecomprising SEQ ID NO:19 or SEQ ID NO:21 and a second polypeptidecomprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQID NO:30, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, or SEQ ID NO:48, wherein the first polypeptide isdirectly linked to the second polypeptide. In some embodiments, thebinding agent comprises a first polypeptide comprising SEQ ID NO:19 orSEQ ID NO:21 and a second polypeptide comprising SEQ ID NO:30 or SEQ IDNO:31, wherein the first polypeptide is directly linked to the secondpolypeptide.

In some embodiments, the binding agent comprises a first polypeptidecomprising SEQ ID NO:19 or SEQ ID NO:21 and a second polypeptidecomprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQID NO:30, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, or SEQ ID NO:48, wherein the first polypeptide isconnected to the second polypeptide by a linker. In some embodiments,the binding agent comprises a first polypeptide comprising SEQ ID NO:19or SEQ ID NO:21 and a second polypeptide comprising SEQ ID NO:30 or SEQID NO:31, wherein the first polypeptide is connected to the secondpolypeptide by a linker.

In some embodiments, the binding agent comprises a first polypeptidethat is at least 80% identical to SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:38, and a secondpolypeptide comprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48, wherein the firstpolypeptide is directly linked to the second polypeptide. In someembodiments, the first polypeptide is at least 85%, at least 90%, atleast 95% identical to SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, or SEQ ID NO:38.

In some embodiments, the binding agent comprises a first polypeptidethat is at least 80% identical to SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:38 and a secondpolypeptide comprising SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48, wherein the firstpolypeptide is connected to the second polypeptide by a linker. In someembodiments, the first polypeptide is at least 85%, at least 90%, atleast 95% identical to SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, or SEQ ID NO:38.

Receptor proteins generally contain a signal sequence that directs thetransport of the proteins. Signal sequences (also referred to as signalpeptides or leader sequences) are located at the N-terminus of nascentpolypeptides. They target the polypeptide to the endoplasmic reticulumand the proteins are sorted to their destinations, for example, to theinner space of an organelle, to an interior membrane, to the cell outermembrane, or to the cell exterior via secretion. Most signal sequencesare cleaved from the protein by a signal peptidase after the proteinsare transported to the endoplasmic reticulum. The cleavage of the signalsequence from the polypeptide usually occurs at a specific site in theamino acid sequence and is dependent upon amino acid residues within thesignal sequence. Although there is usually one specific cleavage site,more than one cleavage site may be recognized and/or used by a signalpeptidase resulting in a non-homogenous N-terminus of the polypeptide.For example, the use of different cleavage sites within a signalsequence can result in a polypeptide expressed with different N-terminalamino acids. Accordingly, in some embodiments, the polypeptides asdescribed herein may comprise a mixture of polypeptides with differentN-termini. In some embodiments, the N-termini differ in length by 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, theN-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In someembodiments, the polypeptide is substantially homogeneous, i.e., thepolypeptides have the same N-terminus. In some embodiments, the signalsequence of the polypeptide comprises one or more (e.g., one, two,three, four, five, six, seven, eight, nine, ten, etc.) amino acidsubstitutions and/or deletions. In some embodiments, the signal sequenceof the polypeptide comprises amino acid substitutions and/or deletionsthat allow one cleavage site to be dominant, thereby resulting in asubstantially homogeneous polypeptide with one N-terminus. In someembodiments, the signal sequence of the polypeptide is not a native(e.g., PVR family member) signal sequence.

Those skilled in the art will appreciate that some of the binding agentsof this invention will comprise fusion proteins in which at least aportion of the Fc region has been deleted or otherwise altered so as toprovide desired biochemical characteristics, such as reduced serumhalf-life, increased serum half-life, or increased target celllocalization, when compared with a fusion protein of approximately thesame immunogenicity comprising a native or unaltered Fc region.Modifications to the Fc region may include additions, deletions, orsubstitutions of one or more amino acids in one or more domains. Themodified fusion proteins disclosed herein may comprise alterations ormodifications to one or more of the two heavy chain constant domains(CH2 or CH3) or to the hinge region. In other embodiments, the entireCH2 domain may be removed (ΔCH2 constructs). In some embodiments, theomitted constant region domain is replaced by a short amino acid spacer(e.g., 10 aa residues) that provides some of the molecular flexibilitytypically imparted by the absent constant region domain.

In some embodiments, the modified fusion protein is engineered to linkthe CH3 domain directly to the hinge region or to the first polypeptide.In other embodiments, a peptide spacer is inserted between the hingeregion of the first polypeptide and the modified CH2 and/or CH3 domains.For example, constructs may be expressed wherein the CH2 domain has beendeleted and the remaining CH3 domain (modified or unmodified) is joinedto the hinge region or first polypeptide with a 5-20 amino acid spacer.Such a spacer may be added to ensure that the regulatory elements of theconstant domain remain free and accessible or that the hinge regionremains flexible. However, it should be noted that amino acid spacersmay, in some cases, prove to be immunogenic and elicit an unwantedimmune response against the construct. Accordingly, in certainembodiments, any spacer added to the construct will be relativelynon-immunogenic so as to maintain the desired biological qualities ofthe fusion protein.

In some embodiments, the modified fusion protein may have only a partialdeletion of a constant domain or substitution of a few or even a singleamino acid. For example, the mutation of a single amino acid in selectedareas of the CH2 domain may be enough to substantially reduce Fc bindingand thereby increase target cell localization. Similarly, it may bedesirable to simply delete the part of one or more constant regiondomains that control a specific effector function (e.g., complement C1qbinding). Such partial deletions of the constant regions may improveselected characteristics of the binding agent (e.g., serum half-life)while leaving other desirable functions associated with the subjectconstant region domain intact. Moreover, as alluded to above, theconstant regions of the disclosed fusion proteins may be modifiedthrough the mutation or substitution of one or more amino acids thatenhances the profile of the resulting construct. In this respect it maybe possible to disrupt the activity provided by a conserved binding site(e.g., Fc binding) while substantially maintaining the configuration andimmunogenic profile of the modified fusion protein. In certainembodiments, the modified fusion protein comprises the addition of oneor more amino acids to the constant region to enhance desirablecharacteristics such as decreasing or increasing effector function, orprovides for more cytotoxin or carbohydrate attachment sites.

It is known in the art that the constant region mediates severaleffector functions. For example, binding of the C1 component ofcomplement to the Fc region of IgG or IgM antibodies (bound to antigen)activates the complement system. Activation of complement is importantin the opsonization and lysis of cell pathogens. The activation ofcomplement also stimulates the inflammatory response and can also beinvolved in autoimmune hypersensitivity. In addition, the Fc region canbind to a cell expressing a Fc receptor (FcR). There are a number of Fcreceptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors).

Thus, in some embodiments, the modified fusion protein provides foraltered effector functions that, in turn, affect the biological profileof the administered agent. For example, in some embodiments, thedeletion or inactivation (through point mutations or other means) of aconstant region domain may reduce Fc receptor binding of the circulatingmodified agent, thereby increasing target cell localization. In otherembodiments, the constant region modifications increase or reduce theserum half-life of the agent. In some embodiments, the constant regionis modified to eliminate disulfide linkages or oligosaccharideattachment sites.

In certain embodiments, a modified fusion protein does not have one ormore effector functions normally associated with an Fc region. In someembodiments, the agent has no ADCC activity, and/or no CDC activity. Incertain embodiments, the agent does not bind to a Fc receptor and/orcomplement factors. In certain embodiments, the agent has no effectorfunction.

This invention also encompasses heterodimeric molecules. Generally theheterodimeric molecule comprises two polypeptides. In some embodiments,the heterodimeric molecule is capable of binding at least two targets.The targets may be, for example, two different receptors on a singlecell or two different targets on two separate cells. Thus, in someembodiments, one polypeptide of the heterodimeric molecule comprises apolypeptide described herein (e.g., binds TIGIT) and one polypeptide ofthe heterodimeric molecule is an antibody. In some embodiments, theheterodimeric molecule is capable of binding one target and alsocomprises a “non-binding” function. Thus in some embodiments, onepolypeptide of the heterodimeric molecule comprises a polypeptidedescribed herein (e.g., binds TIGIT) and one polypeptide of theheterodimeric molecule is an immune response stimulating agent. As usedherein, the phrase “immune response stimulating agent” is used in thebroadest sense and refers to a substance that directly or indirectlystimulates the immune system by inducing activation or increasingactivity of any of the immune system's components. For example, immuneresponse stimulating agents include cytokines, as well as variousantigens including tumor antigens, and antigens derived from pathogens.In some embodiments, the immune response stimulating agent includes, butis not limited to, a colony stimulating factor (e.g.,granulocyte-macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF), granulocyte colony stimulating factor(G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3,IL-7, IL-12, IL-15, IL-18), an antibody that blocks immunosuppressivefunctions (e.g., an anti-CTLA4 antibody, anti-CD28 antibody, anti-CD3antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member ofthe B7 family (e.g., CD80, CD86).

In some embodiments, the heterodimeric molecule can bind a first target,(e.g., TIGIT) as well as a second target, such as an effector moleculeon a leukocyte (e.g., CD2, CD3, CD28, or CD80) or a Fc receptor (e.g.,CD64, CD32, or CD16) so as to elicit a stronger cellular immuneresponse.

In some embodiments, a heterodimeric molecule has enhanced potency ascompared to an individual agent. It is known to those of skill in theart that any agent (e.g., a soluble receptor or a cytokine) may haveunique pharmacokinetics (PK) (e.g., circulating half-life). In someembodiments, a heterodimeric molecule has the ability to synchronize thePK of two active agents and/or polypeptides wherein the two individualagents and/or polypeptides have different PK profiles. In someembodiments, a heterodimeric molecule has the ability to concentrate theactions of two agents and/or polypeptides in a common area (e.g., atumor and/or tumor environment). In some embodiments, a heterodimericmolecule has the ability to concentrate the actions of two agents and/orpolypeptides to a common target (e.g., a tumor or a tumor cell). In someembodiments, a heterodimeric molecule has the ability to target theactions of two agents and/or polypeptides to more than one biologicalpathway or more than one aspect of the immune response. In someembodiments, the heterodimeric molecule has decreased toxicity and/orside effects than either of the agents and/or polypeptides alone. Insome embodiments, the heterodimeric molecule has decreased toxicityand/or side effects as compared to a mixture of the two individualagents and/or polypeptides. In some embodiments, the heterodimericmolecule has an increased therapeutic index. In some embodiments, theheterodimeric molecule has an increased therapeutic index as compared toa mixture of the two individual agents and/or polypeptides or the agentsand/or polypeptides as single agents.

In some embodiments, the binding agent is a multidimeric molecule whichcomprises a first CH3 domain and a second CH3 domain, each of which ismodified to promote formation of heteromultimers or heterodimers. Insome embodiments, the first and second CH3 domains are modified using aknobs-into-holes technique. In some embodiments, the first and secondCH3 domains comprise changes in amino acids that result in alteredelectrostatic interactions. In some embodiments, the first and secondCH3 domains comprise changes in amino acids that result in alteredhydrophobic/hydrophilic interactions (see, for example, U.S. Patent App.Publication No. 2011/0123532).

In some embodiments, the binding agent (e.g., soluble receptor orpolypeptide) is a heterodimeric molecule which comprises heavy chainconstant regions selected from the group consisting of: (a) a firsthuman IgG1 constant region, wherein the amino acids at positionscorresponding to positions 253 and 292 of SEQ ID NO:39 are replaced withglutamate or aspartate, and a second human IgG1 constant region, whereinthe amino acids at positions corresponding to 240 and 282 of SEQ IDNO:39 are replaced with lysine; (b) a first human IgG2 constant region,wherein the amino acids at positions corresponding to positions 249 and288 of SEQ ID NO:40 are replaced with glutamate or aspartate, and asecond human IgG2 constant region wherein the amino acids at positionscorresponding to positions 236 and 278 of SEQ ID NO:40 are replaced withlysine; (c) a first human IgG3 constant region, wherein the amino acidsat positions corresponding to positions 300 and 339 of SEQ ID NO:41 arereplaced with glutamate or aspartate, and a second human IgG3 constantregion wherein the amino acids at positions corresponding to positions287 and 329 of SEQ ID NO:41 are replaced with lysine; and (d) a firsthuman IgG4 constant region, wherein the amino acids at positionscorresponding to positions 250 and 289 of SEQ ID NO:42 are replaced withglutamate or aspartate, and a second IgG4 constant region wherein theamino acids at positions corresponding to positions 237 and 279 of SEQID NO:42 are replaced with lysine.

In some embodiments, the heterodimeric protein comprises twopolypeptides, wherein each polypeptide comprises a human IgG2 CH3domain, and wherein the amino acids at positions corresponding topositions 249 and 288 of SEQ ID NO:40 of one IgG2 CH3 domain arereplaced with glutamate or aspartate, and wherein the amino acids atpositions corresponding to positions 236 and 278 of SEQ ID NO:40 of theother IgG2 CH3 domain are replaced with lysine.

In some embodiments, the binding agent (e.g., a soluble receptor) is aheterodimeric molecule which comprises a first human IgG1 constantregion with amino acid substitutions at positions corresponding topositions 253 and 292 of SEQ ID NO:39, wherein the amino acids arereplaced with glutamate or aspartate, and a second human IgG1 constantregion with amino acid substitutions at positions corresponding topositions 240 and 282 of SEQ ID NO:39, wherein the amino acids arereplaced with lysine. In some embodiments, the binding agent (e.g., asoluble receptor) is a fusion protein which comprises a first human IgG2constant region with amino acid substitutions at positions correspondingto positions 249 and 288 of SEQ ID NO:40, wherein the amino acids arereplaced with glutamate or aspartate, and a second human IgG2 constantregion with amino acid substitutions at positions corresponding topositions 236 and 278 of SEQ ID NO:40, wherein the amino acids arereplaced with lysine. In some embodiments, the binding agent (e.g., asoluble receptor) is a fusion protein which comprises a first human IgG3constant region with amino acid substitutions at positions correspondingto positions 300 and 339 of SEQ ID NO:41, wherein the amino acids arereplaced with glutamate or aspartate, and a second human IgG3 constantregion with amino acid substitutions at positions corresponding topositions 287 and 329 of SEQ ID NO:41, wherein the amino acids arereplaced with lysine. In some embodiments, the binding agent (e.g., asoluble receptor) is a fusion protein which comprises a first human IgG4constant region with amino acid substitutions at positions correspondingto positions 250 and 289 of SEQ ID NO:42, wherein the amino acids arereplaced with glutamate or aspartate, and a second human IgG4 constantregion with amino acid substitutions at positions corresponding topositions 237 and 279 of SEQ ID NO:42, wherein the amino acids arereplaced with lysine.

In some embodiments, the binding agent (e.g., a soluble receptor) is afusion protein which comprises a first human IgG2 constant region withamino acid substitutions at positions corresponding to positions 249 and288 of SEQ ID NO:40, wherein the amino acids are replaced withglutamate, and a second human IgG2 constant region with amino acidsubstitutions at positions corresponding to positions 236 and 278,wherein the amino acids are replaced with lysine. In some embodiments,the binding agent (e.g., a soluble receptor) is a fusion protein whichcomprises a first human IgG2 constant region with amino acidsubstitutions at positions corresponding to positions 249 and 288,wherein the amino acids are replaced with aspartate, and a second humanIgG2 constant region with amino acid substitutions at positionscorresponding to positions 236 and 278, wherein the amino acids arereplaced with lysine.

In some embodiments, the binding agents described herein are monovalent.In some embodiments, the binding agent is a heterodimeric protein thatis monovalent. In some embodiments, the binding agent comprises asoluble receptor that is monovalent. In some embodiments, the bindingagents described herein are bivalent. In some embodiments, the bindingagents described herein are monospecific. In some embodiments, thebinding agents described herein are bispecific. In some embodiments, thebinding agents described herein are multispecific.

The some embodiments, the binding agents are substantially homologous tothe soluble receptors and/or polypeptides described herein. Thesebinding agents can contain, for example, conservative substitutionmutations, i.e. the substitution of one or more amino acids by similaramino acids. For example, conservative substitution refers to thesubstitution of an amino acid with another within the same general classsuch as, for example, one acidic amino acid with another acidic aminoacid, one basic amino acid with another basic amino acid, or one neutralamino acid by another neutral amino acid. What is intended by aconservative amino acid substitution is well known in the art anddescribed herein.

In some embodiments, the binding agents are bispecific antibodies.Bispecific antibodies are capable of specifically recognizing andbinding at least two different epitopes. The different epitopes caneither be within the same molecule (e.g., two epitopes on human TIGIT)or on different molecules (e.g., one epitope on TIGIT and one epitope onCD96). In some embodiments, the bispecific antibodies are monoclonalhuman or humanized antibodies. In some embodiments, the antibodies canspecifically recognize and bind a first antigen target, (e.g., TIGIT) aswell as a second antigen target, such as an effector molecule on aleukocyte (e.g., CD2, CD3, CD28, or CD80) or a Fc receptor (e.g., CD64,CD32, or CD16) so as to focus cellular defense mechanisms to the cellexpressing the first antigen target. In some embodiments, the antibodiescan be used to direct cytotoxic agents to cells which express aparticular target antigen. These antibodies possess an antigen-bindingarm and an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA.

Techniques for making bispecific antibodies are known by those skilledin the art, see for example, Millstein et al., 1983, Nature,305:537-539; Brennan et al., 1985, Science, 229:81; Suresh et al., 1986,Methods in Enzymol., 121:120; Traunecker et al., 1991, EMBO J.,10:3655-3659; Shalaby et al., 1992, J. Exp. Med., 175:217-225; Kostelnyet al., 1992, J. Immunol., 148:1547-1553; Gruber et al., 1994, J.Immunol., 152:5368; U.S. Pat. No. 5,731,168; and U.S. Patent PublicationNo. 2011/0123532). Bispecific antibodies can be intact antibodies orantibody fragments. Antibodies with more than two valencies are alsocontemplated. For example, trispecific antibodies can be prepared (Tuttet al., 1991, J. Immunol., 147:60).

In some embodiments, the binding agent is a bispecific antibody thatspecifically binds the extracellular domain of human TIGIT. In someembodiments, the bispecific antibody specifically binds theextracellular domain of TIGIT and the extracellular domain of CD96. Insome embodiments, the binding agent is a bispecific antibody comprisinga first antigen-binding site that specifically binds human TIGIT and asecond antigen-binding site that specifically binds human CD96. In someembodiments, the binding agent is a bispecific antibody comprising afirst antigen-binding site that specifically binds human TIGIT and asecond antigen-binding site that specifically binds human CD96, whereinthe light chains of the first and second antigen-binding sites areidentical.

In some embodiments, the binding agent is a bispecific antibody thatspecifically binds the extracellular domain of human TIGIT and blockssignaling of TIGIT. In some embodiments, the binding agent is abispecific antibody that specifically binds the extracellular domain ofhuman TIGIT and binds the extracellular domain of human CD96 and blockssignaling of TIGIT and block signaling of CD96.

The binding agents (e.g., soluble receptors or polypeptides) of thepresent invention can be assayed for specific binding by any methodknown in the art. The immunoassays which can be used include, but arenot limited to, competitive and non-competitive assay systems usingtechniques such as Biacore analysis, FACS analysis, immunofluorescence,immunocytochemistry, Western blots, radioimmunoassays, ELISA, “sandwich”immunoassays, immunoprecipitation assays, precipitation reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, and protein A immunoassays. Such assays areroutine and well-known in the art (see, e.g., Ausubel et al., Editors,1994-present, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., New York, N.Y.).

For example, the specific binding of a binding agent (e.g., a solublereceptor) to a target such as TIGIT may be determined using ELISA. AnELISA assay comprises preparing antigen, coating wells of a 96 wellmicrotiter plate with antigen, adding the binding agent conjugated to adetectable compound such as an enzymatic substrate (e.g. horseradishperoxidase or alkaline phosphatase) to the well, incubating for a periodof time and detecting the presence of the binding agent bound to theantigen. In some embodiments, the binding agent is not conjugated to adetectable compound, but instead an antibody conjugated to a detectablecompound that recognizes the binding agent (e.g., PE-conjugated anti-Fcantibody) is added to the well. In some embodiments, instead of coatingthe well with the antigen, the binding agent can be coated to the welland an antibody conjugated to a detectable compound can be addedfollowing the addition of the antigen to the coated well. One of skillin the art would be knowledgeable as to the parameters that can bemodified to increase the signal detected as well as other variations ofELISAs known in the art.

In another example, the specific binding of a binding agent (e.g., asoluble receptor) to a target may be determined using FACS. A FACSscreening assay may comprise generating a cDNA construct that expressesan antigen as a fusion protein (e.g., TIGIT-CD4TM), transfecting theconstruct into cells, expressing the antigen on the surface of thecells, mixing the binding agent with the transfected cells, andincubating for a period of time. The cells bound by the binding agentmay be identified by using a secondary antibody conjugated to adetectable compound that recognizes the binding agent (e.g.,PE-conjugated anti-Fc antibody) and a flow cytometer. A FACS screeningassay may be used to identify a binding agent that binds more than onereceptor, for example, TIGIT and CD96. A FACS screening assay may beused to show that a binding agent does not bind a receptor or bindsweakly to a receptor. One of skill in the art would be knowledgeable asto the parameters that can be modified to optimize the signal detectedas well as other variations of FACS that may enhance screening (e.g.,screening for blocking agents).

The binding affinity of a binding agent (e.g., a soluble receptor) to atarget (e.g., TIGIT) and the off-rate of a binding agent/targetinteraction can be determined by competitive binding assays. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled target (e.g., ³H or ¹²⁵I), or fragment or variantthereof, with the binding agent of interest in the presence ofincreasing amounts of unlabeled target followed by the detection of thebinding agent bound to the labeled target. The affinity of the bindingagent for a target (e.g., TIGIT) and the binding off-rates can bedetermined from the data by Scatchard plot analysis. In someembodiments, Biacore kinetic analysis is used to determine the bindingon and off rates of binding agents that bind a target (e.g., TIGIT).Biacore kinetic analysis comprises analyzing the binding anddissociation of binding agents from chips with immobilized target (e.g.,TIGIT) on the chip surface.

In some embodiments, the binding agent (e.g., a soluble receptor) bindsTIGIT with a dissociation constant (K_(D)) of about 1 μM or less, about100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM orless, about 1 nM or less, or about 0.1 nM or less. In some embodiments,the binding agent binds TIGIT with a K_(D) of about 1 nM or less. Insome embodiments, the binding agent binds TIGIT with a K_(D) of about0.1 nM or less. In some embodiments, the binding agent binds human TIGITwith a K_(D) of about 0.1 nM or less. In some embodiments, the bindingagent (e.g., a soluble receptor) also binds CD96 with a K_(D) of about 1μM or less, about 100 nM or less, about 40 nM or less, about 20 nM orless, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.In some embodiments, the binding agent also binds CD96 with a K_(D) ofabout 1 nM or less. In some embodiments, the binding agent also bindsCD96 with a K_(D) of about 0.1 nM or less. In some embodiments, thebinding agent also binds CD96 with a K_(D) of about 0.1 nM or less. Insome embodiments, the binding agent binds both human TIGIT and mouseTIGIT with a K_(D) of about 10 nM or less. In some embodiments, thebinding agent binds both human TIGIT and mouse TIGIT with a K_(D) ofabout 1 nM or less. In some embodiments, the binding agent binds bothhuman TIGIT and mouse TIGIT with a K_(D) of about 0.1 nM or less. Insome embodiments, the binding agent does not bind human CD226. In someembodiments, the binding agent binds human CD226 with a high K_(D) (weakbinding).

In some embodiments, the dissociation constant of the binding agent toTIGIT is the dissociation constant determined using a TIGIT fusionprotein comprising at least a portion of the TIGIT extracellular domainimmobilized on a Biacore chip. In some embodiments, the dissociationconstant of the binding agent to CD96 is the dissociation constantdetermined using a CD96 fusion protein comprising at least a portion ofthe CD96 extracellular domain immobilized on a Biacore chip. In someembodiments, the dissociation constant of the binding agent or lack ofbinding to CD226 is the dissociation constant determined using a CD226fusion protein comprising at least a portion of the CD226 extracellulardomain immobilized on a Biacore chip.

In some embodiments, the binding agent binds human TIGIT with a halfmaximal effective concentration (EC₅₀) of about 1 μM or less, about 100nM or less, about 40 nM or less, about 20 nM or less, about 10 nM orless, about 1 nM or less, or about 0.1 nM or less. In certainembodiments, the binding agent also binds human CD96 with an EC₅₀ ofabout 40 nM or less, about 20 nM or less, about 10 nM or less, about 1nM or less or about 0.1 nM or less.

In certain embodiments, the binding agents described herein bind TIGITand/or CD96 and modulate an immune response. In some embodiments, abinding agent (e.g., a soluble receptor) activates and/or increases animmune response. In some embodiments, a binding agent increases,promotes, or enhances cell-mediated immunity. In some embodiments, abinding agent increases, promotes, or enhances innate cell-mediatedimmunity. In some embodiments, a binding agent increases, promotes, orenhances adaptive cell-mediated immunity. In some embodiments, a bindingagent increases, promotes, or enhances T-cell activity. In someembodiments, a binding agent increases, promotes, or enhances cytolyticT-cell (CTL) activity. In some embodiments, a binding agent increases,promotes, or enhances NK cell activity. In some embodiments, a bindingagent increases, promotes, or enhances lymphokine-activated killer cell(LAK) activity. In some embodiments, a binding agent increases,promotes, or enhances tumor cell killing. In some embodiments, a bindingagent increases, promotes, or enhances the inhibition of tumor growth.

In some embodiments, the binding agents described herein bind TIGIT andinhibit TIGIT signaling. In some embodiments, a binding agent (e.g., asoluble receptor) binds TIGIT and blocks TIGIT signaling. In someembodiments, a binding agent is an antagonist of TIGIT-mediatedsignaling. In some embodiments, the binding agents described herein bindCD96 and inhibit CD96 signaling. In some embodiments, a binding agent(e.g., a soluble receptor) binds CD96 and blocks CD96 signaling. In someembodiments, a binding agent is an antagonist of CD96-mediatedsignaling. In some embodiments, the binding agents described herein bindTIGIT and CD96 and inhibit TIGIT signaling and CD96 signaling. In someembodiments, a binding agent (e.g., a soluble receptor) binds TIGIT andCD96 and blocks TIGIT signaling and blocks CD96 signaling. In someembodiments, a binding agent is an antagonist of TIGIT-mediatedsignaling and an antagonist of CD96-mediated signaling. In someembodiments, the binding agents described herein bind TIGIT and inhibitTIGIT signaling, but do not bind (or bind weakly to) CD226 and do notinhibit CD226 signaling. In some embodiments, the binding agentsdescribed herein bind TIGIT and CD96 and inhibit TIGIT and CD96signaling, but do not bind (or bind weakly to) CD226 and do not inhibitCD226 signaling. In some embodiments, the binding agents describedherein bind TIGIT, inhibit TIGIT signaling, and increase CD226signaling. In some embodiments, the binding agents described herein bindTIGIT and CD96, inhibit TIGIT and CD96 signaling, and increase CD226signaling. In some embodiments, the binding agents described hereinincrease CD226 signaling.

In some embodiments, a binding agent comprises a soluble receptorcomprising a PVR variant described herein, wherein the PVR variant bindsTIGIT and blocks TIGIT activity. In some embodiments, a binding agentcomprises a soluble receptor comprising a PVR variant described herein,wherein the PVR variant binds TIGIT and blocks TIGIT activity and alsobinds CD96 and blocks CD96 activity. In some embodiments, a bindingagent comprises a soluble receptor comprising a PVR variant describedherein, wherein the PVR variant binds TIGIT and increases CD226activity.

In certain embodiments, a binding agent described herein is an agonist(either directly or indirectly) of human CD226. In some embodiments, thebinding agent is an agonist of CD226 and activates and/or increases animmune response. In some embodiments, the binding agent is an agonist ofCD226 and activates and/or increases activity of NK cells and/or T-cells(e.g., cytolytic activity or cytokine production). In certainembodiments, the binding agent increases the activity by at least about10%, at least about 20%, at least about 30%, at least about 50%, atleast about 75%, at least about 90%, or about 100%.

In certain embodiments, a binding agent described herein is anantagonist (either directly or indirectly) of TIGIT and/or CD96. In someembodiments, the binding agent is an antagonist of TIGIT and/or CD96 andactivates and/or increases an immune response. In some embodiments, thebinding agent is an antagonist of TIGIT and/or CD96 and activates and/orincreases activity of NK cells and/or T-cells (e.g., cytolytic activityor cytokine production). In certain embodiments, the binding agent thebinding agent increases the activity by at least about 10%, at leastabout 20%, at least about 30%, at least about 50%, at least about 75%,at least about 90%, or about 100%.

In certain embodiments, a binding agent described herein increasesactivation of a NK cell. In certain embodiments, a binding agent (e.g.,soluble receptor) increases activation of a T-cell. In certainembodiments, the activation of a NK cell and/or a T-cell by an bindingagent results in an increase in the level of activation of a NK celland/or a T-cell of at least about 10%, at least about 25%, at leastabout 50%, at least about 75%, at least about 90%, or at least about95%.

In vivo and in vitro assays for determining whether a binding agent (orcandidate binding agent) modulates an immune response are known in theart or are being developed. In some embodiments, a functional assay thatdetects T-cell activation may be used. For example, a population ofT-cells can be stimulated with irradiated allogeneic cells expressingPVR, in the presence or absence of a binding agent described herein. Anagent that blocks TIGIT and/or CD96 signaling will cause an increase inthe T-cell activation, as measured by proliferation and cell cycleprogression, IL-2 production, and/or up-regulation of CD25 and CD69. Insome embodiments, a functional assay that detects NK cell activity maybe used. For example, a population of target cells expressing PVR can beco-cultured with NK cells, in the presence or absence of a binding agentdescribed herein. An agent that blocks TIGIT and/or CD96 signaling willcause an increase in the percentage of target cells killed by the NKcells.

In certain embodiments, the binding agents are capable of inhibitingtumor growth. In certain embodiments, the binding agents are capable ofinhibiting tumor growth in vivo (e.g., in a xenograft mouse model,and/or in a human having cancer).

In certain embodiments, the binding agents are capable of reducing thetumorigenicity of a tumor. In certain embodiments, the binding agent iscapable of reducing the tumorigenicity of a tumor in an animal model,such as a mouse xenograft model. In certain embodiments, the bindingagent is capable of reducing the tumorigenicity of a tumor comprisingcancer stem cells in an animal model, such as a mouse xenograft model.In certain embodiments, the number or frequency of cancer stem cells ina tumor is reduced by at least about two-fold, about three-fold, aboutfive-fold, about ten-fold, about 50-fold, about 100-fold, or about1000-fold. In certain embodiments, the reduction in the number orfrequency of cancer stem cells is determined by limiting dilution assayusing an animal model. Additional examples and guidance regarding theuse of limiting dilution assays to determine a reduction in the numberor frequency of cancer stem cells in a tumor can be found, e.g., inInternational Publication Number WO 2008/042236; U.S. Patent PublicationNo. 2008/0064049; and U.S. Patent Publication No. 2008/0178305.

In certain embodiments, the binding agents have one or more of thefollowing effects: inhibit proliferation of tumor cells, inhibit tumorgrowth, reduce the tumorigenicity of a tumor, reduce the tumorigenicityof a tumor by reducing the frequency of cancer stem cells in the tumor,trigger cell death of tumor cells, increase cell contact-dependentgrowth inhibition, increase tumor cell apoptosis, reduce epithelialmesenchymal transition (EMT), or decrease survival of tumor cells. Insome embodiments, the binding agents have one or more of the followingeffects: inhibit viral infection, inhibit chronic viral infection,reduce viral load, trigger cell death of virus-infected cells, or reducethe number or percentage of virus-infected cells.

In certain embodiments, the binding agents described herein have acirculating half-life in mice, cynomolgus monkeys, or humans of at leastabout 5 hours, at least about 10 hours, at least about 24 hours, atleast about 3 days, at least about 1 week, or at least about 2 weeks. Incertain embodiments, the binding agent is an IgG (e.g., IgG1 or IgG2)fusion protein that has a circulating half-life in mice, cynomolgusmonkeys, or humans of at least about 5 hours, at least about 10 hours,at least about 24 hours, at least about 3 days, at least about 1 week,or at least about 2 weeks. Methods of increasing (or decreasing) thehalf-life of agents such as polypeptides and soluble receptors are knownin the art. For example, known methods of increasing the circulatinghalf-life of IgG fusion proteins include the introduction of mutationsin the Fc region which increase the pH-dependent binding of the antibodyto the neonatal Fc receptor (FcRn) at pH 6.0 (see, e.g., U.S. PatentPublication Nos. 2005/0276799, 2007/0148164, and 2007/0122403). Knownmethods of increasing the circulating half-life of soluble receptorslacking a Fc region include such techniques as PEGylation.

In some embodiments of the present invention, the binding agents arepolypeptides. The polypeptides can be recombinant polypeptides, naturalpolypeptides, or synthetic polypeptides that bind TIGIT and/or CD96. Itwill be recognized in the art that some amino acid sequences of theinvention can be varied without significant effect of the structure orfunction of the protein. Thus, the invention further includes variationsof the polypeptides which show substantial binding activity to TIGITand/or CD96. In some embodiments, amino acid sequence variations of thepolypeptides include deletions, insertions, inversions, repeats, and/orother types of substitutions.

The polypeptides, analogs and variants thereof, can be further modifiedto contain additional chemical moieties not normally part of thepolypeptide. The derivatized moieties can improve the solubility, thebiological half-life, and/or absorption of the polypeptide. The moietiescan also reduce or eliminate undesirable side effects of thepolypeptides and variants. An overview for chemical moieties can befound in Remington: The Science and Practice of Pharmacy, 22^(st)Edition, 2012, Pharmaceutical Press, London.

The polypeptides described herein can be produced by any suitable methodknown in the art. Such methods range from direct protein synthesismethods to constructing a DNA sequence encoding polypeptide sequencesand expressing those sequences in a suitable host. In some embodiments,a DNA sequence is constructed using recombinant technology by isolatingor synthesizing a DNA sequence encoding a wild-type protein of interest.Optionally, the sequence can be mutagenized by site-specific mutagenesisto provide functional analogs thereof. See, e.g., Zoeller et al., 1984,PNAS, 81:5662-5066 and U.S. Pat. No. 4,588,585.

In some embodiments, a DNA sequence encoding a polypeptide of interestmay be constructed by chemical synthesis using an oligonucleotidesynthesizer. Oligonucleotides can be designed based on the amino acidsequence of the desired polypeptide and selecting those codons that arefavored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizea polynucleotide sequence encoding an isolated polypeptide of interest.For example, a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular isolated polypeptide can besynthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or anothermethod), the polynucleotide sequences encoding a particular polypeptideof interest can be inserted into an expression vector and operativelylinked to an expression control sequence appropriate for expression ofthe protein in a desired host. Proper assembly can be confirmed bynucleotide sequencing, restriction enzyme mapping, and/or expression ofa biologically active polypeptide in a suitable host. As is well-knownin the art, in order to obtain high expression levels of a transfectedgene in a host, the gene must be operatively linked to transcriptionaland translational expression control sequences that are functional inthe chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding the binding agents (e.g., solublereceptors) described herein. For example, recombinant expression vectorscan be replicable DNA constructs which have synthetic or cDNA-derivedDNA fragments encoding a polypeptide chain of a binding agentoperatively linked to suitable transcriptional and/or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences. Regulatory elements can include an operatorsequence to control transcription. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants can additionally beincorporated. DNA regions are “operatively linked” when they arefunctionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. In some embodiments,structural elements intended for use in yeast expression systems includea leader sequence enabling extracellular secretion of translated proteinby a host cell. In other embodiments, where recombinant protein isexpressed without a leader or transport sequence, it can include anN-terminal methionine residue. This residue can optionally besubsequently cleaved from the expressed recombinant protein to provide afinal product.

The choice of an expression control sequence and an expression vectordepends upon the choice of host. A wide variety of expressionhost/vector combinations can be employed. Useful expression vectors foreukaryotic hosts include, for example, vectors comprising expressioncontrol sequences from SV40, bovine papilloma virus, adenovirus, andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, including pCR1,pBR322, pMB9 and their derivatives, and wider host range plasmids, suchas M13 and other filamentous single-stranded DNA phages.

Suitable host cells for expression of a polypeptide (or a protein to useas a target) include prokaryotes, yeast cells, insect cells, or highereukaryotic cells under the control of appropriate promoters. Prokaryotesinclude gram-negative or gram-positive organisms, for example E. coli orBacillus. Higher eukaryotic cells include established cell lines ofmammalian origin as described below. Cell-free translation systems mayalso be employed. Appropriate cloning and expression vectors for usewith bacterial, fungal, yeast, and mammalian cellular hosts aredescribed by Pouwels et al. (1985, Cloning Vectors: A Laboratory Manual,Elsevier, New York, N.Y.). Additional information regarding methods ofprotein production, including antibody production, can be found, e.g.,in U.S. Patent Publication No. 2008/0187954; U.S. Pat. Nos. 6,413,746and 6,660,501; and International Patent Publication No. WO 2004/009823.

Various mammalian cell culture systems are used to express recombinantpolypeptides. Expression of recombinant proteins in mammalian cells canbe preferred because such proteins are generally correctly folded,appropriately modified, and biologically functional. Examples ofsuitable mammalian host cell lines include COS-7 (monkeykidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammarytumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamsterovary-derived), HeLa (human cervical cancer-derived), BHK (hamsterkidney fibroblast-derived), and HEK-293 (human embryonic kidney-derived)cell lines and variants thereof. Mammalian expression vectors cancomprise non-transcribed elements such as an origin of replication, asuitable promoter and enhancer linked to the gene to be expressed, andother 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′non-translated sequences, such as necessary ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

Expression of recombinant proteins in insect cell culture systems (e.g.,baculovirus) also offers a robust method for producing correctly foldedand biologically functional proteins. Baculovirus systems for productionof heterologous proteins in insect cells are well-known to those ofskill in the art (see, e.g., Luckow and Summers, 1988, Bio/Technology,6:47).

Thus, the present invention provides cells comprising the binding agentsdescribed herein. In some embodiments, the cells produce the bindingagents described herein. In certain embodiments, the cells produce afusion protein. In some embodiments, the cells produce a solublereceptor. In some embodiments, the cells produce an antibody. In someembodiments, the cells produce a bispecific antibody. In someembodiments, the cells produce a heterodimeric protein.

The proteins produced by a transformed host can be purified according toany suitable method. Standard methods include chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for proteinpurification Affinity tags such as hexa-histidine, maltose bindingdomain, influenza coat sequence, and glutathione-S-transferase can beattached to the protein to allow easy purification by passage over anappropriate affinity column. Isolated proteins can also be physicallycharacterized using such techniques as proteolysis, mass spectrometry(MS), nuclear magnetic resonance (NMR), high performance liquidchromatography (HPLC), and x-ray crystallography.

In some embodiments, supernatants from expression systems which secreterecombinant protein into culture media can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. In some embodiments, an anion exchange resin can beemployed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose, or other types commonly employed in proteinpurification. In some embodiments, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. In some embodiments, ahydroxyapatite media can be employed, including but not limited to,ceramic hydroxyapatite (CHT). In certain embodiments, one or morereverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a binding agent. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a homogeneous recombinant protein.

In some embodiments, recombinant protein produced in bacterial culturecan be isolated, for example, by initial extraction from cell pellets,followed by one or more concentration, salting-out, aqueous ionexchange, or size exclusion chromatography steps. HPLC can be employedfor final purification steps. Microbial cells employed in expression ofa recombinant protein can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents.

Methods known in the art for purifying polypeptides also include, forexample, those described in U.S. Patent Publication Nos. 2008/0312425,2008/0177048, and 2009/0187005.

In certain embodiments, a binding agent described herein is apolypeptide that does not comprise an immunoglobulin Fc region. Incertain embodiments, the polypeptide comprises a protein scaffold of atype selected from the group consisting of protein A, protein G, alipocalin, a fibronectin domain, an ankyrin consensus repeat domain, andthioredoxin. A variety of methods for identifying and producingnon-antibody polypeptides that bind with high affinity to a proteintarget are known in the art. See, e.g., Skerra, 2007, Curr. Opin.Biotechnol., 18:295-304; Hosse et al., 2006, Protein Science, 15:14-27;Gill et al., 2006, Curr. Opin. Biotechnol., 17:653-658; Nygren, 2008,FEBS J., 275:2668-76; and Skerra, 2008, FEBS J., 275:2677-83. In certainembodiments, phage display technology may be used to produce and/oridentify a binding polypeptide. In certain embodiments, mammalian celldisplay technology may be used to produce and/or identify a bindingpolypeptide.

It can further be desirable to modify a polypeptide in order to increase(or decrease) its serum half-life. This can be achieved, for example, byincorporation of a salvage receptor binding epitope into the polypeptideby mutation of the appropriate region in the polypeptide or byincorporating the epitope into a peptide tag that is then fused to thepolypeptide at either end or in the middle (e.g., by DNA or peptidesynthesis).

Heteroconjugate molecules are also within the scope of the presentinvention. Heteroconjugate molecules are composed of two covalentlyjoined polypeptides. Such molecules have, for example, been proposed totarget immune cells to unwanted cells, such as tumor cells. It is alsocontemplated that the heteroconjugate molecules can be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate.

In certain embodiments, a binding agent described herein can be used inany one of a number of conjugated (i.e. an immunoconjugate orradioconjugate) or non-conjugated forms. In certain embodiments, thebinding agents can be used in a non-conjugated form to harness thesubject's natural defense mechanisms including CDC and ADCC to eliminatemalignant or cancer cells.

In certain embodiments, a binding agent described herein is a smallmolecule. The term “small molecule” generally refers to a low molecularweight organic compound which is by definition not a peptide/protein. Asmall molecule binding agent described herein may bind to TIGIT and/orCD96 with high affinity and interfere with or block the interaction ofTIGIT and/or CD96 with PVR. In some embodiments, the small moleculeinterferes with or blocks the interaction of TIGIT and/or CD96 with PVR,disrupting TIGIT signaling, but does not disrupt CD226 signaling.

In some embodiments, a binding agent described herein is conjugated to acytotoxic agent. In some embodiments, the cytotoxic agent is achemotherapeutic agent including, but not limited to, methotrexate,adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents. In some embodiments, thecytotoxic agent is an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof, including, but notlimited to, diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes. In some embodiments, the cytotoxic agent is aradioisotope to produce a radioconjugate or a radioconjugated bindingagent. A variety of radionuclides are available for the production ofradioconjugated binding agents including, but not limited to, ⁹⁰Y, ¹²⁵I,¹³¹I, ¹²³I, ¹¹¹In, ¹³¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, and ²¹²Bi. Conjugates of a binding agent and one or more smallmolecule toxins, such as a calicheamicin, maytansinoids, a trichothene,and CC1065, and the derivatives of these toxins that have toxinactivity, can also be used. In some embodiments, a binding agentdescribed herein is conjugated to a maytansinoid. In some embodiments, abinding agent described herein is conjugated to mertansine (DM1).Conjugates of a binding agent and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

III. Polynucleotides

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a binding agent (e.g., a solublereceptor or polypeptide) described herein. The term “polynucleotidesthat encode a polypeptide” encompasses a polynucleotide which includesonly coding sequences for the polypeptide as well as a polynucleotidewhich includes additional coding and/or non-coding sequences. Thepolynucleotides of the invention can be in the form of RNA or in theform of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and canbe double-stranded or single-stranded, and if single stranded can be thecoding strand or non-coding (anti-sense) strand.

In certain embodiments, the polynucleotide comprises a polynucleotideencoding a polypeptide comprising an amino acid sequence selected fromthe group consisting of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQID NO:38. In certain embodiments, the polynucleotide comprises apolynucleotide encoding a polypeptide comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20, SEQ ID NO:21, and SEQ ID NO:38.

In certain embodiments, a polynucleotide comprises a polynucleotidehaving a nucleotide sequence at least 80% identical, at least 85%identical, at least 90% identical, at least 95% identical, and in someembodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotideencoding an amino acid sequence selected from the group consisting of:SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, and SEQ ID NO:38. In certainembodiments, a polynucleotide comprises a polynucleotide having anucleotide sequence at least 80% identical, at least 85% identical, atleast 90% identical, at least 95% identical, and in some embodiments, atleast 96%, 97%, 98% or 99% identical to a polynucleotide encoding anamino acid sequence selected from the group consisting of: SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:38. Alsoprovided is a polynucleotide that comprises a polynucleotide thathybridizes to a polynucleotide encoding an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,and SEQ ID NO:38. Also provided is a polynucleotide that comprises apolynucleotide that hybridizes to a polynucleotide encoding an aminoacid sequence selected from the group consisting of: SEQ ID NO:18, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:38. Also provided isa polynucleotide that comprises a polynucleotide that hybridizes to thecomplement of a polynucleotide encoding an amino acid sequence selectedfrom the group consisting of: SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, and SEQ ID NO:38. In certain embodiments, thehybridization is under conditions of high stringency. Conditions of highstringency are known to those of skill in the art and may include butare not limited to, (1) employ low ionic strength and high temperaturefor washing, for example 15 mM sodium chloride/1.5 mM sodium citrate(1×SSC) with 0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 in 5×SSC(0.75M NaCl, 75 mM sodium citrate) at 42° C.; or (3) employ 50%formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes in0.2×SSC containing 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

In certain embodiments, a polynucleotide comprises the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g., a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments, a polynucleotide comprises the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g., COS-7cells) is used. In some embodiments, the marker sequence is a FLAG-tag,a peptide of sequence DYKDDDDK (SEQ ID NO:32) which can be used inconjunction with other affinity tags.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs,and/or derivatives.

In certain embodiments, the present invention provides a polynucleotidecomprising a polynucleotide having a nucleotide sequence at least about80% identical, at least about 85% identical, at least about 90%identical, at least about 95% identical, and in some embodiments, atleast about 96%, 97%, 98% or 99% identical to a polynucleotide encodinga polypeptide comprising a binding agent (e.g., a soluble receptor or apolypeptide) described herein.

As used herein, the phrase a polynucleotide having a nucleotide sequenceat least, for example, 95% “identical” to a reference nucleotidesequence is intended to mean that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence can include up to five point mutations per each100 nucleotides of the reference nucleotide sequence. In other words, toobtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence can be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence can be inserted into the referencesequence. These mutations of the reference sequence can occur at the 5′or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among nucleotides in the reference sequence or in one ormore contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments, apolynucleotide variant contains alterations which produce silentsubstitutions, additions, or deletions, but does not alter theproperties or activities of the encoded polypeptide. In someembodiments, a polynucleotide variant comprises silent substitutionsthat results in no change to the amino acid sequence of the polypeptide(due to the degeneracy of the genetic code). Polynucleotide variants canbe produced for a variety of reasons, for example, to optimize codonexpression for a particular host (i.e., change codons in the human mRNAto those preferred by a bacterial host such as E. coli). In someembodiments, a polynucleotide variant comprises at least one silentmutation in a non-coding or a coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate oralter expression (or expression levels) of the encoded polypeptide. Insome embodiments, a polynucleotide variant is produced to increaseexpression of the encoded polypeptide. In some embodiments, apolynucleotide variant is produced to decrease expression of the encodedpolypeptide. In some embodiments, a polynucleotide variant has increasedexpression of the encoded polypeptide as compared to a parentalpolynucleotide sequence. In some embodiments, a polynucleotide varianthas decreased expression of the encoded polypeptide as compared to aparental polynucleotide sequence.

In some embodiments, at least one polynucleotide variant is produced(without changing the amino acid sequence of the encoded polypeptide) toincrease production of a heterodimeric molecule. In some embodiments, atleast one polynucleotide variant is produced (without changing the aminoacid sequence of the encoded polypeptide) to increase production of abispecific antibody.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure.

Vectors and cells comprising the polynucleotides described herein arealso provided. In some embodiments, an expression vector comprises apolynucleotide molecule. In some embodiments, a host cell comprises anexpression vector comprising the polynucleotide molecule. In someembodiments, a host cell comprises a polynucleotide molecule.

IV. Methods of Use and Pharmaceutical Compositions

The binding agents of the invention are useful in a variety ofapplications including, but not limited to, therapeutic treatmentmethods, such as immunotherapy for cancer. In certain embodiments, thebinding agents are useful for activating, promoting, increasing, and/orenhancing an immune response, inhibiting tumor growth, reducing tumorvolume, increasing tumor cell apoptosis, and/or reducing thetumorigenicity of a tumor. The binding agents of the invention are alsouseful for immunotherapy against pathogens, such as viruses. In certainembodiments, the binding agents are useful for activating, promoting,increasing, and/or enhancing an immune response, inhibiting viralinfection, reducing viral infection, increasing virally-infected cellapoptosis, and/or increasing killing of virus-infected cells. Themethods of use may be in vitro, ex vivo, or in vivo methods. In someembodiments, a binding agent is an agonist of an immune response. Insome embodiments, a binding agent is an antagonist of TIGIT. In someembodiments, a binding agent is an antagonist of CD96. In someembodiments, a binding agent is an antagonist of TIGIT and CD96. In someembodiments, a binding agent is an agonist of CD226.

The present invention provides methods for activating an immune responsein a subject using the binding agents described herein. In someembodiments, the invention provides methods for promoting an immuneresponse in a subject using a binding agent described herein. In someembodiments, the invention provides methods for increasing an immuneresponse in a subject using a binding agent described herein. In someembodiments, the invention provides methods for enhancing an immuneresponse in a subject using a binding agent described herein. In someembodiments, the activating, promoting, increasing, and/or enhancing ofan immune response comprises increasing cell-mediated immunity. In someembodiments, the activating, promoting, increasing, and/or enhancing ofan immune response comprises increasing T-cell activity. In someembodiments, the activating, promoting, increasing, and/or enhancing ofan immune response comprises increasing CTL activity. In someembodiments, the activating, promoting, increasing, and/or enhancing ofan immune response comprises increasing NK cell activity. In someembodiments, the activating, promoting, increasing, and/or enhancing ofan immune response comprises increasing T-cell activity and increasingNK cell activity. In some embodiments, the activating, promoting,increasing, and/or enhancing of an immune response comprises increasingCU activity and increasing NK cell activity. In some embodiments, theimmune response is a result of antigenic stimulation. In someembodiments, the antigenic stimulation is a tumor cell. In someembodiments, the antigenic stimulation is cancer. In some embodiments,the antigenic stimulation is a pathogen. In some embodiments, theantigenic stimulation is a virally-infected cell.

In some embodiments, a method of increasing an immune response in asubject comprises administering to the subject a therapeuticallyeffective amount of a binding agent described herein, wherein thebinding agent inhibits the interaction between TIGIT and PVR, inhibitsthe interaction between CD96 and PVR, and does not inhibit theinteraction between CD226 and PVR.

In some embodiments, the invention provides methods of increasing theactivity of CD226-positive cells. In some embodiments, the methodcomprises contacting the CD226-positive cells with an effective amountof a binding agent described herein. In some embodiments, theCD226-positive cells are T-cells, NK cells, monocytes, macrophages,and/or B-cells. In some embodiments, the increasing of activity ofCD226-positive cells is evidenced by increased cytolytic activity. Insome embodiments, the increasing of activity of CD226-positive cells isevidenced by increased killing of target cells. In some embodiments, theincreasing of activity of CD226-positive cells is evidenced by increasedkilling of tumor cells. In some embodiments, the increasing of activityof CD226-positive cells is evidenced by inhibition of tumor growth. Insome embodiments, the increasing of activity of CD226-positive cells isevidenced by inhibition of viral infection. In some embodiments, theincreasing of activity of CD226-positive cells is evidenced by increasedkilling of virally-infected cells.

The present invention also provides methods for inhibiting growth of atumor using the binding agents described herein. In certain embodiments,the method of inhibiting growth of a tumor comprises contacting a cellmixture with a binding agent in vitro. For example, an immortalized cellline or a cancer cell line mixed with immune cells (e.g., T-cells or NKcells) is cultured in medium to which is added a binding agent. In someembodiments, tumor cells are isolated from a patient sample such as, forexample, a tissue biopsy, pleural effusion, or blood sample, mixed withimmune cells (e.g., T-cells and/or NK cells), and cultured in medium towhich is added a binding agent. In some embodiments, the binding agentincreases, promotes, and/or enhances the activity of the immune cells.In some embodiments, the binding agent inhibits tumor cell growth. Insome embodiments, the binding agent comprises a soluble receptor. Insome embodiments, the binding agent is a soluble receptor. In someembodiments, the binding agent is an antibody. In some embodiments, thebinding agent is a polypeptide.

In some embodiments, the method of inhibiting growth of a tumorcomprises contacting the tumor or tumor cells with a binding agent invivo. In certain embodiments, contacting a tumor or tumor cell with abinding agent is undertaken in an animal model. For example, a bindingagent may be administered to mice which have syngeneic tumors. In someembodiments, the binding agent increases, promotes, and/or enhances theactivity of immune cells in the mice. In some embodiments, the bindingagent inhibits tumor growth. In some embodiments, the binding agent isadministered at the same time or shortly after introduction of tumorcells into the animal to prevent tumor growth (“preventative model”). Insome embodiments, the binding agent is administered as a therapeuticafter tumors have grown to a specified size (“therapeutic model”). Insome embodiments, the binding agent comprises a soluble receptor. Insome embodiments, the binding agent is a soluble receptor. In someembodiments, the binding agent is an antibody. In some embodiments, thebinding agent is a polypeptide.

In certain embodiments, the method of inhibiting growth of a tumorcomprises administering to a subject a therapeutically effective amountof a binding agent described herein. In certain embodiments, the subjectis a human. In certain embodiments, the subject has a tumor or has had atumor which was removed. In some embodiments, the binding agentcomprises a soluble receptor. In some embodiments, the binding agent isa soluble receptor. In some embodiments, the binding agent is anantibody. In some embodiments, the binding agent is a polypeptide.

In addition, the invention provides a method of inhibiting growth of atumor in a subject, comprising administering a therapeutically effectiveamount of a binding agent to the subject. In certain embodiments, thetumor comprises cancer stem cells. In certain embodiments, the frequencyof cancer stem cells in the tumor is reduced by administration of thebinding agent. In some embodiments, a method of reducing the frequencyof cancer stem cells in a tumor in a subject, comprising administeringto the subject a therapeutically effective amount of a binding agent isprovided. In some embodiments, the binding agent comprises a solublereceptor. In some embodiments, the binding agent is a soluble receptor.In some embodiments, the binding agent is an antibody. In someembodiments, the binding agent is a polypeptide.

In some embodiments, a method of inhibiting tumor growth in a subjectcomprises: administering to the subject a therapeutically effectiveamount of a binding agent described herein, wherein the binding agentinhibits the interaction between TIGIT and PVR, inhibits the interactionbetween CD96 and PVR, and does not inhibit the interaction between CD226and PVR. In some embodiments, the PVR is expressed on the tumor cell. Insome embodiments, TIGIT is expressed on NK cells and/or T-cells. In someembodiments, CD96 is expressed on NK cells and/or T-cells. In someembodiments, CD226 is expressed on NK cells and/or T-cells. In someembodiments, PVR is expressed on tumor cells and TIGIT and CD226 areexpressed on NK cells and/or T-cells. In some embodiments, PVR isexpressed on tumor cells and TIGIT, CD96, and CD226 are expressed on NKcells and/or T-cells.

In addition, the invention provides a method of reducing thetumorigenicity of a tumor in a subject, comprising administering to asubject a therapeutically effective amount of a binding agent describedherein. In certain embodiments, the tumor comprises cancer stem cells.In some embodiments, the tumorigenicity of a tumor is reduced byreducing the frequency of cancer stem cells in the tumor. In someembodiments, the methods comprise using the binding agents describedherein. In certain embodiments, the frequency of cancer stem cells inthe tumor is reduced by administration of a binding agent.

In some embodiments, the tumor is a solid tumor. In certain embodiments,the tumor is a tumor selected from the group consisting of: colorectaltumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breasttumor, kidney tumor, prostate tumor, neuroendocrine tumor,gastrointestinal tumor, melanoma, cervical tumor, bladder tumor,glioblastoma, and head and neck tumor. In certain embodiments, the tumoris a colorectal tumor. In certain embodiments, the tumor is an ovariantumor. In some embodiments, the tumor is a lung tumor. In certainembodiments, the tumor is a pancreatic tumor. In certain embodiments,the tumor is a melanoma tumor.

The present invention further provides methods for treating cancer in asubject comprising administering a therapeutically effective amount ofthe binding agent to a subject. In some embodiments, the binding agentbinds the extracellular domain of TIGIT and/or CD96, increases an immuneresponse, and inhibits or reduces growth of the cancer. In someembodiments, the binding agent binds TIGIT. In some embodiments, thebinding agent binds TIGIT and CD96. In some embodiments, the bindingagent binds TIGIT and does not bind (or binds weakly to) CD226. In someembodiments, the binding agent binds TIGIT and CD96 and does not bind(or binds weakly to) CD226. In some embodiments, the binding agentcomprises a soluble receptor. In some embodiments, the binding agent isa soluble receptor. In some embodiments, the binding agent is anantibody. In some embodiments, the binding agent is a polypeptide.

The present invention provides for methods of treating cancer comprisingadministering a therapeutically effective amount of a binding agentdescribed herein to a subject (e.g., a subject in need of treatment). Incertain embodiments, the subject is a human. In certain embodiments, thesubject has a cancerous tumor. In certain embodiments, the subject hashad a tumor removed.

In certain embodiments, the cancer is a cancer selected from the groupconsisting of colorectal cancer, pancreatic cancer, lung cancer, ovariancancer, liver cancer, breast cancer, kidney cancer, prostate cancer,gastrointestinal cancer, melanoma, cervical cancer, neuroendocrinecancer, bladder cancer, glioblastoma, and head and neck cancer. Incertain embodiments, the cancer is pancreatic cancer. In certainembodiments, the cancer is ovarian cancer. In certain embodiments, thecancer is colorectal cancer. In certain embodiments, the cancer isbreast cancer. In certain embodiments, the cancer is prostate cancer. Incertain embodiments, the cancer is lung cancer. In certain embodiments,the cancer is melanoma.

In some embodiments, the cancer is a hematologic cancer. In someembodiment, the cancer is selected from the group consisting of: acutemyelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cellacute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia(CLL), hairy cell leukemia, chronic myelogenous leukemia (CML),non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).

The invention also provides a method of inactivating, inhibiting, orsuppressing TIGIT and/or CD96 signaling in a cell comprising contactingthe cell with an effective amount of a binding agent described herein.In certain embodiments, the cell is a T-cell. In some embodiments, thecell is a cytolytic cell. In some embodiments, the cell is a CTL. Insome embodiments, the cell is a NK cell. In certain embodiments, themethod is an in vivo method wherein the step of contacting the cell withthe binding agent comprises administering a therapeutically effectiveamount of the binding agent to the subject. In some embodiments, themethod is an in vitro or ex vivo method. In certain embodiments, thebinding agent inhibits, suppresses, and/or decreases TIGIT and/or CD96signaling. In some embodiments, the binding agent comprises a solublereceptor. In some embodiments, the binding agent is a soluble receptor.In some embodiments, the binding agent is a polypeptide. In someembodiments, the binding agent is an antibody.

The invention also provides a method of activating or enhancing CD226signaling in a cell comprising contacting the cell with an effectiveamount of a binding agent described herein. In certain embodiments, thecell is a T-cell. In some embodiments, the cell is a cytolytic cell. Insome embodiments, the cell is a CTL. In some embodiments, the cell is aNK cell. In certain embodiments, the method is an in vivo method whereinthe step of contacting the cell with the binding agent comprisesadministering a therapeutically effective amount of the binding agent tothe subject. In some embodiments, the method is an in vitro or ex vivomethod. In certain embodiments, the binding agent activates, promotes,induces, enhances, and/or increases CD226 signaling. In someembodiments, the binding agent comprises a soluble receptor. In someembodiments, the binding agent is a soluble receptor. In someembodiments, the binding agent is a polypeptide. In some embodiments,the binding agent is an antibody.

Over-expression or aberrant exposure of some members of theimmunoglobulin superfamily on cells (e.g., tumor cells or virallyinfected cells) may allow the receptors to serve as targets forsurveillance by the immune system (“immunosurveillance”). For example, acentral characteristic of epithelial cell biology is that epithelialcells exist in single-cell layers. As such, they have three distinctsurfaces, an apical surface exposed to the lumen, a basolateral membranethat interacts with the basement membrane, and an “intercellularsurface” forming the interaction region between adjacent cells. Withoutbeing bound by theory, we believe that some of the members of the Igsuperfamily would generally be restricted to this third surface, theintercellular surface, as this would be the likely region to enabledirect cell-cell communication.

Many proteins are involved in cell-to-cell interactions and cellinteractions with the microenvironment. Some of these proteins are knownto reside within the intercellular membrane region, including cadherenswhich contribute to adherens junctions, connexins which contribute togap junctions, and claudins and occludin which contribute to tightjunctions. In addition to these proteins, other proteins are thought toreside in the apical junctional complex created by the tight junctionsand adherens junctions. For example, within some normal cellulararchitecture members of the Ig superfamily (e.g., receptors) would beexpressed at the intercellular surfaces and would not be detected by abinding agent described herein. However, a cell with altered cellularmorphology or a cell that has lost normal cellular architecture (e.g., atumor cell or a virally-infected cell) may have aberrant exposure of aprotein/receptor, for example, PVR, PVRL2, and/or PVRL3, making thesecells detectable by surveillance with the binding agents describedherein.

In addition, over-expression of a PVR family member on a cell's surfacemay make that cell a better target to cells expressing counter receptors(e.g., CTLs and/or NK cells). Interestingly, human PVR and PVRL2 havebeen found to be over-expressed on certain tumors, including colorectalcancer, gastric cancers, ovarian cancers, neuroblastomas, myeloidleukemias, and multiple myeloma (see, for example, Masson et al., 2001,Gut, 49:236-240; Tahara-Hanaoka et al., 2006, Blood, 107:1491-1496;Carlsten et al., 2007, Cancer Res., 67:1317-1325; Castriconi et al.,2004, Cancer Res., 64:9180-9184; Pende et al., 2005, Blood,105:2066-2073; El-Sherbiny et al., 2007, Cancer Res., 67:8444-8449).

Thus, the present invention provides methods of identifying a humansubject for treatment with a binding agent, comprising determining ifthe subject has a tumor that has an elevated level of PVR as compared toexpression of PVR in a reference sample or a pre-determined level ofPVR. As used herein, a “reference sample” includes but is not limitedto, normal tissue, non-cancerous tissue of the same tissue type, tumortissue of the same tissue type, and tumor tissue of a different tissuetype. Thus, in some embodiments, the level of expression of PVR in atumor is compared to the level of expression of PVR in normal tissue. Insome embodiments, the level of expression of PVR in a tumor is comparedto the level of expression of PVR in non-cancerous tissue of the sametissue type. In some embodiments, the level of expression of PVR in atumor is compared to the level of expression of PVR in tumors of thesame tissue type. In some embodiments, the level of expression of PVR ina tumor is compared to the level of expression of PVR in tumors of adifferent tissue type. In some embodiments, the level of expression ofPVR in a tumor is compared to a pre-determined level of PVR. In someembodiments, determining the level of PVR expression is done prior totreatment. In some embodiments, determining the level of PVR expressionis by immunohistochemistry. In some embodiments, the subject isadministered a binding agent described herein if the tumor has anelevated level of PVR expression as compared to the expression of PVR inthe reference sample or the pre-determined level. For example, in someembodiments, the subject is administered a binding agent describedherein if the tumor has an elevated level of PVR expression as comparedto the level of PVR expression in a reference sample. In someembodiments, the subject is administered a binding agent describedherein if the tumor has an elevated level of PVR expression as comparedto a pre-determined level of PVR.

In some embodiments, if the tumor has an elevated level of PVR, thesubject is selected for treatment with a binding agent that specificallybinds TIGIT and/or CD96. In some embodiments, if selected for treatment,the subject is administered a binding agent described herein. In certainembodiments, the subject has had a tumor removed.

The present invention also provides methods of identifying a humansubject for treatment with a binding agent, comprising determining ifthe subject has a tumor that has an aberrant expression of PVR ascompared to expression of PVR in tissue of the same type or in areference sample. In some embodiments, if the tumor has an aberrantexpression of PVR, the subject is selected for treatment with a bindingagent that specifically binds TIGIT and/or CD96. In some embodiments, ifselected for treatment, the subject is administered a binding agentdescribed herein. In certain embodiments, the subject has had a tumorremoved.

The present invention also provides methods of selecting a human subjectfor treatment with a binding agent described herein, the methodcomprising determining if the subject has a tumor that has an elevatedexpression level of PVR, wherein if the tumor has an elevated expressionlevel of PVR the subject is selected for treatment. In some embodiments,a method of inhibiting tumor growth in a human subject comprisesdetermining if the tumor has an elevated expression level of PVR, andadministering to the subject a therapeutically effective amount of abinding agent described herein. In some embodiments, a method oftreating cancer in a human subject comprises (a) selecting a subject fortreatment based, at least in part, on the subject having a cancer thathas an elevated level of PVR, and (b) administering to the subject atherapeutically effective amount of a binding agent described herein.

Methods for determining the level of PVR nucleic acid expression in acell, tumor, or cancer are known by those of skill in the art. Thesemethods include, but are not limited to, PCR-based assays, microarrayanalyses, and nucleotide sequencing (e.g., NextGen sequencing). Methodsfor determining the level of PVR protein expression in a cell, tumor, orcancer include, but are not limited to, Western blot analysis, proteinarrays, ELISAs, immunohistochemistry (IHC), and FACS.

Methods for determining whether a tumor or cancer has an elevated levelof PVR expression can use a variety of samples. In some embodiments, thesample is taken from a subject having a tumor or cancer. In someembodiments, the sample is a fresh tumor/cancer sample. In someembodiments, the sample is a frozen tumor/cancer sample. In someembodiments, the sample is a formalin-fixed paraffin-embedded sample. Insome embodiments, the sample is a blood sample. In some embodiments, thesample is a plasma sample. In some embodiments, the sample is processedto a cell lysate. In some embodiments, the sample is processed to DNA orRNA.

The present invention further provides pharmaceutical compositionscomprising the binding agents described herein. In certain embodiments,the pharmaceutical compositions further comprise a pharmaceuticallyacceptable vehicle. In some embodiments, the pharmaceutical compositionsfind use in immunotherapy. In some embodiments, the pharmaceuticalcompositions find use in inhibiting tumor growth in a subject (e.g., ahuman patient). In some embodiments, the pharmaceutical compositionsfind use in treating cancer in a subject (e.g., a human patient).

In certain embodiments, formulations are prepared for storage and use bycombining a purified binding agent of the present invention with apharmaceutically acceptable vehicle (e.g., a carrier or excipient).Suitable pharmaceutically acceptable vehicles include, but are notlimited to, nontoxic buffers such as phosphate, citrate, and otherorganic acids; salts such as sodium chloride; antioxidants includingascorbic acid and methionine; preservatives such asoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride, benzethonium chloride, phenol, butyl or benzylalcohol, alkyl parabens, such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weightpolypeptides (e.g., less than about 10 amino acid residues); proteinssuch as serum albumin, gelatin, or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; carbohydrates such asmonosaccharides, disaccharides, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes suchas Zn-protein complexes; and non-ionic surfactants such as TWEEN orpolyethylene glycol (PEG). (Remington: The Science and Practice ofPharmacy, 22^(st) Edition, 2012, Pharmaceutical Press, London.).

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical by epidermal or transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders; pulmonary by inhalation or insufflation of powdersor aerosols, including by nebulizer, intratracheal, and intranasal;oral; or parenteral including intravenous, intraarterial, intratumoral,subcutaneous, intraperitoneal, intramuscular (e.g., injection orinfusion), or intracranial (e.g., intrathecal or intraventricular).

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, orsuppositories. In solid compositions such as tablets the principalactive ingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, anddiluents (e.g., water). These can be used to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention, or a non-toxic pharmaceutically acceptable saltthereof. The solid preformulation composition is then subdivided intounit dosage forms of a type described above. The tablets, pills, etc. ofthe formulation or composition can be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, the tablet or pill can comprise an inner composition covered byan outer component. Furthermore, the two components can be separated byan enteric layer that serves to resist disintegration and permits theinner component to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials include a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The binding agents described herein can also be entrapped inmicrocapsules. Such microcapsules are prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions asdescribed in Remington: The Science and Practice of Pharmacy, 22^(st)Edition, 2012, Pharmaceutical Press, London.

In certain embodiments, pharmaceutical formulations include a bindingagent of the present invention complexed with liposomes. Methods toproduce liposomes are known to those of skill in the art. For example,some liposomes can be generated by reverse phase evaporation with alipid composition comprising phosphatidylcholine, cholesterol, andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can beextruded through filters of defined pore size to yield liposomes withthe desired diameter.

In certain embodiments, sustained-release preparations can be produced.Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing abinding agent, where the matrices are in the form of shaped articles(e.g., films or microcapsules). Examples of sustained-release matricesinclude polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate)or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

In certain embodiments, in addition to administering a binding agent,the method or treatment further comprises administering at least oneimmune response stimulating agent. In some embodiments, the immuneresponse stimulating agent includes, but is not limited to, a colonystimulating factor (e.g., granulocyte-macrophage colony stimulatingfactor (GM-CSF), macrophage colony stimulating factor (M-CSF),granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)),an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), anantibody that blocks immunosuppressive functions (e.g., an anti-CTLA4antibody, anti-CD28 antibody, anti-CD3 antibody), a toll-like receptor(e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80,CD86). An immune response stimulating agent can be administered priorto, concurrently with, and/or subsequently to, administration of thebinding agent. Pharmaceutical compositions comprising a binding agentand the immune response stimulating agent(s) are also provided. In someembodiments, the immune response stimulating agent comprises 1, 2, 3, ormore immune response stimulating agents.

In certain embodiments, in addition to administering a binding agent,the method or treatment further comprises administering at least oneadditional therapeutic agent. An additional therapeutic agent can beadministered prior to, concurrently with, and/or subsequently to,administration of the binding agent. Pharmaceutical compositionscomprising a binding agent and the additional therapeutic agent(s) arealso provided. In some embodiments, the at least one additionaltherapeutic agent comprises 1, 2, 3, or more additional therapeuticagents.

Combination therapy with two or more therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing toxic side effects and/or increasing the therapeuticindex of the agent(s). Combination therapy may decrease the likelihoodthat resistant cancer cells will develop. In some embodiments,combination therapy comprises a therapeutic agent that affects theimmune response (e.g., enhances or activates the response) and atherapeutic agent that affects (e.g., inhibits or kills) thetumor/cancer cells.

In some embodiments, the combination of a binding agent and at least oneadditional therapeutic agent results in additive or synergistic results.In some embodiments, the combination therapy results in an increase inthe therapeutic index of the binding agent. In some embodiments, thecombination therapy results in an increase in the therapeutic index ofthe additional agent(s). In some embodiments, the combination therapyresults in a decrease in the toxicity and/or side effects of the bindingagent. In some embodiments, the combination therapy results in adecrease in the toxicity and/or side effects of the additional agent(s).

Useful classes of therapeutic agents include, for example, antitubulinagents, auristatins, DNA minor groove binders, DNA replicationinhibitors, alkylating agents (e.g., platinum complexes such ascisplatin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, purine antimetabolites, puromycins, radiation sensitizers,steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or thelike. In certain embodiments, the second therapeutic agent is analkylating agent, an antimetabolite, an antimitotic, a topoisomeraseinhibitor, or an angiogenesis inhibitor.

Therapeutic agents that may be administered in combination with thebinding agents described herein include chemotherapeutic agents. Thus,in some embodiments, the method or treatment involves the administrationof a binding agent of the present invention in combination with achemotherapeutic agent or in combination with a cocktail ofchemotherapeutic agents. Treatment with a binding agent can occur priorto, concurrently with, or subsequent to administration ofchemotherapies. Combined administration can include co-administration,either in a single pharmaceutical formulation or using separateformulations, or consecutive administration in either order butgenerally within a time period such that all active agents can exerttheir biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in The Chemotherapy Source Book, 4^(th) Edition, 2008, M.C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Chemotherapeutic agents useful in the instant invention include, but arenot limited to, alkylating agents such as thiotepa and cyclosphosphamide(CYTOXAN); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine,floxuridine, 5-FU; androgens such as calusterone, dromostanolonepropionate, epitiostanol, mepitiostane, testolactone; anti-adrenals suchas aminoglutethimide, mitotane, trilostane; folic acid replenishers suchas folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel(TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine (XELODA); and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above. In certain embodiments, theadditional therapeutic agent is cisplatin. In certain embodiments, theadditional therapeutic agent is carboplatin.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D,etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well aspharmaceutically acceptable salts, acids, or derivatives of any ofthese. In some embodiments, the additional therapeutic agent isirinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these. In certain embodiments, the additional therapeutic agent isgemcitabine.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. In someembodiments, the agent is a taxane. In certain embodiments, the agent ispaclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, orderivative of paclitaxel or docetaxel. In certain embodiments, the agentis paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel(ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternativeembodiments, the antimitotic agent comprises a vinca alkaloid, such asvincristine, binblastine, vinorelbine, or vindesine, or pharmaceuticallyacceptable salts, acids, or derivatives thereof. In some embodiments,the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor ofa mitotic kinase such as Aurora A or Plk1. In certain embodiments, theadditional therapeutic agent is paclitaxel.

In some embodiments, an additional therapeutic agent comprises an agentsuch as a small molecule. For example, treatment can involve thecombined administration of a binding agent of the present invention witha small molecule that acts as an inhibitor against tumor-associatedantigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.In some embodiments, a binding agent of the present invention isadministered in combination with a protein kinase inhibitor selectedfrom the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA),sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033,cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). Insome embodiments, an additional therapeutic agent comprises an mTORinhibitor.

In certain embodiments, the additional therapeutic agent is a smallmolecule that inhibits a cancer stem cell pathway. In some embodiments,the additional therapeutic agent is an inhibitor of the Notch pathway.In some embodiments, the additional therapeutic agent is an inhibitor ofthe Wnt pathway. In some embodiments, the additional therapeutic agentis an inhibitor of the BMP pathway. In some embodiments, the additionaltherapeutic agent is an inhibitor of the Hippo pathway. In someembodiments, the additional therapeutic agent is an inhibitor of themTOR/AKR pathway.

In some embodiments, an additional therapeutic agent comprises abiological molecule, such as an antibody. For example, treatment caninvolve the combined administration of a binding agent of the presentinvention with antibodies against tumor-associated antigens including,but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF.In certain embodiments, the additional therapeutic agent is an antibodyspecific for a cancer stem cell marker. In some embodiments, theadditional therapeutic agent is an antibody that binds a component ofthe Notch pathway. In some embodiments, the additional therapeutic agentis an antibody that binds a component of the Wnt pathway. In certainembodiments, the additional therapeutic agent is an antibody thatinhibits a cancer stem cell pathway. In some embodiments, the additionaltherapeutic agent is an inhibitor of the Notch pathway. In someembodiments, the additional therapeutic agent is an inhibitor of the Wntpathway. In some embodiments, the additional therapeutic agent is aninhibitor of the BMP pathway. In some embodiments, the additionaltherapeutic agent is an antibody that inhibits β-catenin signaling. Incertain embodiments, the additional therapeutic agent is an antibodythat is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptorantibody). In certain embodiments, the additional therapeutic agent isbevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab(OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, orcetuximab (ERBITUX).

Furthermore, treatment with a binding agent described herein can includecombination treatment with other biologic molecules, such as one or morecytokines (e.g., lymphokines, interleukins, tumor necrosis factors,and/or growth factors) or can be accompanied by surgical removal oftumors, removal of cancer cells, or any other therapy deemed necessaryby a treating physician.

In some embodiments, the binding agent can be combined with a growthfactor selected from the group consisting of, but not limited to:adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin(EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF,migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins,PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, PlGF, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

In certain embodiments, the treatment involves the administration of abinding agent of the present invention in combination with radiationtherapy. Treatment with a binding agent can occur prior to, concurrentlywith, or subsequent to administration of radiation therapy. Dosingschedules for such radiation therapy can be determined by the skilledmedical practitioner.

In certain embodiments, the treatment involves the administration of abinding agent of the present invention in combination with anti-viraltherapy. Treatment with a binding agent can occur prior to, concurrentlywith, or subsequent to administration of antiviral therapy. Theanti-viral drug used in combination therapy will depend upon the virusthe subject is infected with.

Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously.

It will be appreciated that the combination of a binding agent and atleast one additional therapeutic agent may be administered in any orderor concurrently. In some embodiments, the binding agent will beadministered to patients that have previously undergone treatment with asecond therapeutic agent. In certain other embodiments, the bindingagent and a second therapeutic agent will be administered substantiallysimultaneously or concurrently. For example, a subject may be given abinding agent (e.g., a soluble receptor) while undergoing a course oftreatment with a second therapeutic agent (e.g., chemotherapy). Incertain embodiments, a binding agent will be administered within 1 yearof the treatment with a second therapeutic agent. In certain alternativeembodiments, a binding agent will be administered within 10, 8, 6, 4, or2 months of any treatment with a second therapeutic agent. In certainother embodiments, a binding agent will be administered within 4, 3, 2,or 1 weeks of any treatment with a second therapeutic agent. In someembodiments, a binding agent will be administered within 5, 4, 3, 2, or1 days of any treatment with a second therapeutic agent. It will furtherbe appreciated that the two (or more) agents or treatments may beadministered to the subject within a matter of hours or minutes (i.e.,substantially simultaneously).

For the treatment of a disease, the appropriate dosage of a bindingagent of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the binding agent is administered for therapeuticor preventative purposes, previous therapy, the patient's clinicalhistory, and so on, all at the discretion of the treating physician. Thebinding agent can be administered one time or over a series oftreatments lasting from several days to several months, or until a cureis effected or a diminution of the disease state is achieved (e.g.,reduction in tumor size). Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient andwill vary depending on the relative potency of an individual agent. Theadministering physician can determine optimum dosages, dosingmethodologies, and repetition rates. In certain embodiments, dosage isfrom 0.01 μg to 100 mg/kg of body weight, from 0.1 μg to 100 mg/kg ofbody weight, from 1 μg to 100 mg/kg of body weight, from 1 mg to 100mg/kg of body weight, 1 mg to 80 mg/kg of body weight from 10 mg to 100mg/kg of body weight, from 10 mg to 75 mg/kg of body weight, or from 10mg to 50 mg/kg of body weight. In certain embodiments, the dosage of thebinding agent is from about 0.1 mg to about 20 mg/kg of body weight. Insome embodiments, the dosage of the binding agent is about 0.5 mg/kg ofbody weight. In some embodiments, the dosage of the binding agent isabout 1 mg/kg of body weight. In some embodiments, the dosage of thebinding agent is about 1.5 mg/kg of body weight. In some embodiments,the dosage of the binding agent is about 2 mg/kg of body weight. In someembodiments, the dosage of the binding agent is about 2.5 mg/kg of bodyweight. In some embodiments, the dosage of the binding agent is about 5mg/kg of body weight. In some embodiments, the dosage of the bindingagent is about 7.5 mg/kg of body weight. In some embodiments, the dosageof the binding agent is about 10 mg/kg of body weight. In someembodiments, the dosage of the binding agent is about 12.5 mg/kg of bodyweight. In some embodiments, the dosage of the binding agent is about 15mg/kg of body weight. In certain embodiments, the dosage can be givenonce or more daily, weekly, monthly, or yearly. In certain embodiments,the binding agent is given once every week, once every two weeks, onceevery three weeks, or once every four weeks.

In some embodiments, a binding agent may be administered at an initialhigher “loading” dose, followed by one or more lower doses. In someembodiments, the frequency of administration may also change. In someembodiments, a dosing regimen may comprise administering an initialdose, followed by additional doses (or “maintenance” doses) once a week,once every two weeks, once every three weeks, or once every month. Forexample, a dosing regimen may comprise administering an initial loadingdose, followed by a weekly maintenance dose of, for example, one-half ofthe initial dose. Or a dosing regimen may comprise administering aninitial loading dose, followed by maintenance doses of, for exampleone-half of the initial dose every other week. Or a dosing regimen maycomprise administering three initial doses for 3 weeks, followed bymaintenance doses of, for example, the same amount every other week.

As is known to those of skill in the art, administration of anytherapeutic agent may lead to side effects and/or toxicities. In somecases, the side effects and/or toxicities are so severe as to precludeadministration of the particular agent at a therapeutically effectivedose. In some cases, drug therapy must be discontinued, and other agentsmay be tried. However, many agents in the same therapeutic class oftendisplay similar side effects and/or toxicities, meaning that the patienteither has to stop therapy, or if possible, suffer from the unpleasantside effects associated with the therapeutic agent.

Thus, the present invention provides methods of administering to asubject the binding agents described herein comprising using anintermittent dosing strategy for administering one or more agents, whichmay reduce side effects and/or toxicities associated with administrationof a binding agent, chemotherapeutic agent, etc. In some embodiments, amethod for treating cancer in a human subject comprises administering tothe subject a therapeutically effective dose of a binding agent incombination with a therapeutically effective dose of a chemotherapeuticagent, wherein one or both of the agents are administered according toan intermittent dosing strategy. In some embodiments, the intermittentdosing strategy comprises administering an initial dose of a bindingagent to the subject, and administering subsequent doses of the bindingagent about once every 2 weeks. In some embodiments, the intermittentdosing strategy comprises administering an initial dose of a bindingagent to the subject, and administering subsequent doses of the bindingagent about once every 3 weeks. In some embodiments, the intermittentdosing strategy comprises administering an initial dose of a bindingagent to the subject, and administering subsequent doses of the bindingagent about once every 4 weeks. In some embodiments, the binding agentis administered using an intermittent dosing strategy and thechemotherapeutic agent is administered weekly.

V. Screening

The present invention provides screening methods to identify agents thatmodulate the immune response. In some embodiments, the present inventionprovides methods for screening candidate agents, including but notlimited to, proteins, peptides, peptidomimetics, small molecules,compounds, or other drugs, which modulate the immune response.

In some embodiments, a method of screening for a candidate agent thatmodulates the immune response comprises determining if the agent has aneffect on immune response cells. In some embodiments, a method ofscreening for a candidate agent that modulates the immune responsecomprises determining if the agent is capable of increasing the activityof immune cells. In some embodiments, a method of screening for acandidate agent that modulates the immune response comprises determiningif the agent is capable of increasing the activity of cytolytic cells,such as CTLs and/or NK cells.

VI. Kits Comprising Binding Agents

The present invention provides kits that comprise the binding agentsdescribed herein and that can be used to perform the methods describedherein. In certain embodiments, a kit comprises at least one purifiedbinding agent in one or more containers. In some embodiments, the kitscontain all of the components necessary and/or sufficient to perform adetection assay, including all controls, directions for performingassays, and any necessary software for analysis and presentation ofresults. One skilled in the art will readily recognize that thedisclosed binding agents of the present invention can be readilyincorporated into one of the established kit formats which are wellknown in the art.

Further provided are kits that comprise a binding agent as well as atleast one additional therapeutic agent. In certain embodiments, thesecond (or more) therapeutic agent is a chemotherapeutic agent. Incertain embodiments, the second (or more) therapeutic agent is anangiogenesis inhibitor.

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe indetail preparation of certain antibodies of the present disclosure andmethods for using antibodies of the present disclosure. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, may be practiced without departing from the scopeof the present disclosure.

EXAMPLES Example 1 PVR Family Constructs

Protein constructs of PVR family members TIGIT, CD96, CD226, PVRL1,PVRL2, PVRL3, PVRL4, PVR, and PVR variants were prepared includingmembrane-anchored proteins and soluble receptors (FIG. 2). Eachmembrane-anchored receptor was designed to be non-functional in regardto signaling, as the transmembrane and cytoplasmic domains were replacedwith the human CD4 transmembrane domain and an intracellular greenfluorescent protein (GFP) tag. The membrane-anchored protein constructswere generated by ligating at least one domain of the extracellulardomain (ECD) of a human PVR family protein to the transmembrane domainof CD4 and a C-terminal GFP protein tag using standard recombinant DNAtechniques. These constructs are referred to as “PVR familymember”-CD4TM-GFP, for example PVR-CD4TM-GFP. The soluble receptors weredesigned to include at least one domain of the ECD linked to animmunoglobulin Fc domain. The soluble receptor PVR family proteinconstructs were generated by ligating the ECD region of human PVR familymember proteins to the Fc domain of human IgG1 using standardrecombinant DNA techniques. These constructs are referred to as “PVRfamily member”-Fc, for example CD226-Fc. As known to those of skill inthe art, the ECD region of any given protein used in the constructs maycomprise the ECD or comprise a fragment of the ECD, for example just aIgV domain. Also, what is considered to be the ECD or an Ig domain mayvary by one, two, three, or more amino acids at the amino end, thecarboxyl end, or both ends of the domain. These fusion proteins may beused to examine the binding interactions of the PVR family members.

The constructs generated include ECD regions, or a fragment thereof,from the PVR family members in Table 2.

TABLE 2 UniProtKB Name Full name Other names No. SEQ ID NO PVR FamilyPVR Poliovirus receptor NECL-5, P15151 CD155, PVS PVRL1 Poliovirusreceptor-related HVEC, HLGR, Q15223 protein 1 Nectin-1, CD111, PRR1PVRL2 Poliovirus receptor-related HVEB, PRR2, Q92692 protein 2 CD112,Nectin-2 PVRL3 Poliovirus receptor-related Nectin-3, Q9NQS3 protein 3CD113 PVRL4 Poliovirus receptor-related Nectin-4, Q96NY8 protein 4 LNIR,PRR4 CD226 CD226 antigen DNAM1, PTA- Q15762 1, TLiSA1 CD96 T-cellsurface protein tactile P40200 TIGIT T-cell immunoreceptor with IgVSIG9, Vstm3, Q495A1 and ITIM domains WUCAM

Example 2 Binding Interactions Between PVR Family Members

The binding interactions among members of the PVR family were examinedby flow cytometry. Each of the family members was expressed both as anFc fusion protein containing at least one domain of the ECD of thereceptor fused to the Fc region of human IgG1, and also as anmembrane-anchored form containing at least one domain of the ECD of thereceptor fused to a human CD4 transmembrane region and an intracellulargreen fluorescent (GFP) protein tag (see Example 1).

Individual potential binding interactions were assessed by transfectionof HEK-293T cells with an expression vector encoding a specificmembrane-anchored receptor (PVR, PVRL1, PVRL2, PVRL3, or PVRL4), andthen examining the ability of a specific receptor-Fc fusion protein(CD96, TIGIT, or CD226) to bind to the transfected cells. HEK-293T cellswere transiently transfected with a cDNA expression vector encodingPVR-CD4TM-GFP, PVRL1-CD4TM-GFP, PVRL2-CD4TM-GFP, PVRL3-CD4TM-GFP, orPVRL4-CD4TM-GFP and then subsequently mixed with soluble CD226-Fc,TIGIT-Fc, or CD96-Fc fusion proteins. In addition, individual potentialbinding interactions were assessed by transfection of HEK-293T cellswith an expression vector encoding a specific membrane-anchored receptor(PVR, PVRL1, PVRL2, PVRL3, or PVRL4), and then examining the ability ofa specific receptor-Fc fusion protein (PVR, PVRL1, PVRL2, PVRL3, orPVRL4) to bind to the transfected cells. HEK-293T cells were transientlytransfected with a cDNA expression vector encoding PVR-CD4TM-GFP,PVRL1-CD4TM-GFP, PVRL2-CD4TM-GFP, PVRL3-CD4TM-GFP, or PVRL4-CD4TM-GFPand then subsequently mixed with soluble PVR-Fc, PVRL1-Fc, PVRL2-Fc,PVRL3-Fc, or PVRL4-Fc fusion proteins. Binding was detected bysubsequent staining of the cells with an anti-human Fc antibodyconjugated to phycoerythrin (PE) and analysis using flow cytometry.

As shown in FIG. 3A, membrane-anchored PVR was bound by solublereceptors CD226, TIGIT and CD96. In addition, soluble receptor CD226weakly bound to PVRL2, and soluble receptor TIGIT bound to PVRL2, PVRL3,and PVRL4. As shown in FIG. 3B, soluble receptor PVR bound PVRL3,soluble receptor PVRL1 bound PVRL3 and PVRL4; soluble receptor PVRL3bound PVRL1, PVRL2 and PVR; and soluble receptor PVRL4 bound PVRL1.Positive binding interactions are highlighted by circles. Also shown isa schematic representation of the observed binding interactions betweendifferent members of the PVR family (FIG. 3C). Some of the indicatedbinding interactions observed during this analysis appear to be new.

Example 3 Generation of PVR Variants

The crystal structure of PVR bound to TIGIT has been previouslydisclosed (see, Stengel et al., 2012, PNAS, 109:5399-5404). Thestructure was examined and residues within PVR that appeared to not becritical for TIGIT binding, but might potentially impact the binding ofCD226 or CD96 were selected. These residues are highlighted in FIG. 4. AcDNA expression library of variant human PVR N-terminal IgV domainmolecules was designed and generated in which amino acid positions 65,67, 72, 73, 74, 81, 82, 84, and 85 (SEQ ID NO:18) were individuallysubstituted with all twenty amino acids. The cDNA expression vectorencoded the N-terminal IgV domain of PVR fused to a CD4 transmembranedomain and a green fluorescent protein (GFP) tag. The expression vectorplasmid also contained a bacterial ampicillin resistance gene. The cDNAlibrary of variant PVR molecules was transfected into CAP-T cells in thepresence of a 100-fold excess of an irrelevant vector lacking ampicillinresistance. CAP-T cells are an immortalized amniocyte cell line, stablyexpressing the SV40 large T antigen (CEVEC Pharmaceuticals, KolnGermany). This strategy was designed to reduce the number of unique PVRvariant plasmids transfected per cell. Forty-eight hours aftertransfection, cells were incubated with fluorescently-labeled TIGIT-Fc,CD96-Fc, CD226-Fc, a combination of TIGIT-Fc and CD226-Fc, or acombination of CD96-Fc and CD226-Fc. The cells were analyzed byfluorescence activated cell sorting (FACS) to isolate cells thatdisplayed binding to either TIGIT or CD96, but lacked binding to CD226.Plasmids were recovered from the isolated cells, used to transformedbacteria, and the bacteria were plated on ampicillin-containing plates.Plasmids from individual colonies were sequenced and analyzed. In thismanner amino acid substitutions that enable relative binding of PVR toTIGIT, CD96 and CD226 were identified.

FIG. 5 shows the binding pattern of two such amino acid variants. PVRvariant S72N (serine to asparagine) did not significantly impact thebinding to TIGIT or CD96 as compared to wild-type PVR, but the PVRvariant S72N had substantially reduced binding to CD226 as compared towild-type PVR. Another variant, PVR variant Q82K (glutamine to lysine)appeared to have increased the binding to TIGIT compared to wild-typePVR with a different binding pattern in the presence of TIGIT and CD226.This may allow TIGIT to more effectively compete with CD226 for bindingto available variant PVRs.

Example 4 Natural Killer (NK) Cell Cytotoxicity Assays

The human chronic myelogenous leukemia cell line K562 and the human lungadenocarcinoma cell line A549 are cultured in RPMI 1640 culture medium(Gibco/Life Technologies, Carlsbad, Calif.) supplemented with 10% (v/v)fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100μg/ml streptomycin (Gibco) at 37° C. in a humidified atmosphere of 5%CO₂. K562 cells are transfected with GFP, human PVR, or the human PVRvariants (4 μg DNA per 2×10⁶ cells) via electroporation using an AmaxaNucleofector device and Nucleofector Kit V according to themanufacturer's recommendations (Lonza, Basel, Switzerland). Transfectionefficiency is routinely 60-70%, as assessed by flow cytometry for GFPpositivity. A549 cells are transfected with the same constructs (3 μgDNA per 1×10⁶ cells) using FuGENE 6 (Promega, Madison, Wis.) accordingto the manufacturer's instructions. Transfection efficacy is routinely>95%.

Primary human NK cells are isolated directly from fresh peripheral bloodbuffy coats (Stanford Blood Center, Palo Alto, Calif.) by 30-minuteincubation with RosetteSep NK Cell Enrichment Cocktail (Stem CellTechnologies, Vancouver, British Columbia, Canada) prior toFicoll-Hypaque density gradient centrifugation (Stem Cell Technologies).Human NK cells are cultured in L-glutamine-free RPMI 1640 mediumsupplemented with 10% FBS, 100 U/ml of penicillin, and 100 μg/ml ofstreptomycin. Isolated NK cells are routinely >98% CD56+CD3− by flowcytometry. The NK cell line NK-92 was purchased from the American TypeCulture Collection (Manassas, Va.) and is maintained in RPMI 1640containing 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 150U/ml recombinant human IL-2.

NK-92 cells or primary human NK cells are plated in 96-well V-bottomplates with or without 300 U/ml recombinant human IL-2 (PeproTech, RockyHill, N.J.) and incubated overnight at 37° C. In some experiments, NK-92cells or primary NK cells are incubated with 10 μl g/ml of specificblocking antibodies to NKp30, NKp46, or NKG2D (Biolegend, San Diego,Calif.), or an equivalent amount of isotype-matched polyclonal human IgG(Sigma-Aldrich, St. Louis, Mo.) for 30 minutes at 4° C. prior to use incytotoxicity assays. Target cells (K562 or A549 cells transfected withGFP, human PVR, or human PVR variants) are labeled with 10 μM calcein AM(Life Technologies) for 1 hour at 37° C. and then combined with the NKcells at various effector:target ratios (50:1-3:1). Following a 4-hourincubation at 37° C., cell-free supernatants are harvested and calceinrelease is quantified on a fluorometer at an excitation of 485 nm and anemission of 535 nm. The percentage of specific cell lysis is determinedas: % lysis=100×(ER−SR)/(MR−SR), where ER, SR, and MR representexperimental, spontaneous, and maximum calcein release, respectively.Spontaneous release is the fluorescence emitted by target cellsincubated in media alone (i.e., in the absence of effector cells), whilemaximum release is determined by lysing target cells with an equalvolume of 10% SDS.

In some experiments, NK cell cytotoxicity is evaluated in the presenceof soluble human PVR-Fc or the human PVR-Fc variants. Primary human NKcells or NK-92 cells are plated as described above, with the addition of10 μg/ml of PVR-Fc or the human PVR-Fc variants. NK cell lysis againstK562 cells or A549 cells is analyzed as described above.

Freshly isolated primary human NK cells were incubated overnight at 37°C. with or without 300 IU/ml recombinant human IL-2 (PeproTech, RockyHill, N.J.). The NK cells were then pre-treated with 30 μg/ml of PVR-Fcvariant Q82K (gray bar), PVR-Fc wild-type control (black bar), or mediumonly (white bar) for 30 minutes at 4° C. in HBSS. The NK cells werewashed, resuspended in media supplemented with an additional 30 μg/ml ofthe PVR-Fc variant or PVR-Fc WT, and plated in 96-well V-bottom plates.Target cells (HEK-293T cells or K562 cells) were labeled with 10 μMcalcein AM (Life Technologies, Grand Island N.Y.) for 2 hours at 37° C.and then mixed with the NK cells at an effector:target ratio of 12:1.Following a 4-hour incubation at 37° C., cell-free supernatants wereharvested and calcein release was quantified on a fluorometer at anexcitation of 485 nm and an emission of 535 nm. The percentage ofspecific cell lysis was determined as described above.

NK cells demonstrated an increased ability to kill target cells whentreated with PVR variant Q82K as compared to untreated NK cells or NKcells treated with a wild-type PVR (FIG. 6). Cell lysis was increasedwith the addition of IL-2 in all samples.

NK activation and/or activity can also be assessed by measuring theamount of IFN-gamma that is produced by NK cells during an assay. Wellsof a 96-well flat-bottom culture plate were seeded with HEK-293T or A549cells at a density of 5×10⁴ target cells/well. Target cells were grownto confluence overnight. Freshly isolated human NK cells werepre-treated with 30 μg/ml of PVR-Fc variant Q82K (gray bar), PVR-Fcwild-type control (black bar), or medium only (white bar) for 30 minutesat 4° C. in HBSS. NK cells were then washed, resuspended in mediasupplemented with an additional 30 μg/ml of the PVR-Fc variant or PVR-FcWT, and added to the target cells at 2×10⁵ cells/well in mediacontaining 300 IU/ml human IL-2. A duplicate set of cells were set up inmedia without human IL-2. Culture supernatants were harvested after 24hours and analyzed for IFN-gamma content by ELISA (R&D Systems,Minneapolis, Minn.).

In the absence of IL-2, the NK cells produced very limited amounts ofIFN-gamma and there appeared to be little difference between thedifferent samples. In contrast, in the presence of IL-2, the NK cellsproduced higher levels of IFN-gamma when pre-treated with the PVR-Fcvariant Q82K as compared to PVR-Fc wild-type or untreated controls (FIG.7).

Example 5 FACS Analysis of Binding Interactions Between PVR Variants andTIGIT, CD226 and PVRL3

HEK-293T cells were transiently transfected with a cDNA expressionvector encoding PVR-CD4TM-GFP, PVR S72N variant-CD4TM-GFP, PVR Q82Kvariant-CD4TM-GFP, or PVR Q82K+S72N double variant-CD4TM-GFP. After 24hours, cells were mixed with soluble TIGIT-Fc, CD226-Fc or PVRL3-Fcfusion proteins and then subsequently stained with PE-conjugatedanti-human Fc secondary antibody. Fusion protein binding was thenanalyzed by flow cytometry.

The results show that the double mutant PVR fusion protein exhibitsimproved binding to TIGIT as compared to parental wild-type PVR, but nodetectable binding to CD226 (FIGS. 8A and 8B). The PVR variants hadcomparable or somewhat improved binding to PVRL3 relative to parentalwild-type PVR. Therefore, in addition to enhancing an immune response(for example to a tumor), the PVR variants may have the ability tolocalize preferentially to tumors by binding to PVRL3 exposed on tumorsby the disruption of normal tight junction architecture and targetingthe tumor cells for immunosurveillance.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to person skilled in the art and areto be included within the spirit and purview of this application.

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences including both polynucleotide andpolypeptide sequences cited herein are hereby incorporated by referenceherein in their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence were specifically and individuallyindicated to be so incorporated by reference.

The sequences disclosed in the application are:

Human PVR with predicted signal sequence underlined (SEQ ID NO: 1)MARAMAAAWPLLLVALLVLSWPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGMSRNAIIFLVLGILVFLILLGIGIYFYWSKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSSQDPQTEGTRHuman PVRL1 with predicted signal sequence underlined (SEQ ID NO: 2)MARMGLAGAAGRWWGLALGLTAFFLPGVHSQVVQVNDSMYGFIGTDVVLHCSFANPLPSVKITQVTWQKSTNGSKQNVAIYNPSMGVSVLAPYRERVEFLRPSFTDGTIRLSRLELEDEGVYICEFATFPTGNRESQLNLTVMAKPTNWIEGTQAVLRAKKGQDDKVLVATCTSANGKPPSVVSWETRLKGEAEYQEIRNPNGTVTVISRYRLVPSREAHQQSLACIVNYHMDRFKESLTLNVQYEPEVTIEGFDGNWYLQRMDVKLTCKADANPPATEYHWTTLNGSLPKGVEAQNRTLFFKGPINYSLAGTYICEATNPIGTRSGQVEVNITEFPYTPSPPEHGRRAGPVPTAIIGGVAGSILLVLIVVGGIVVALRRRRHTFKGDYSTKKHVYGNGYSKAGIPQHHPPMAQNLQYPDDSDDEKKAGPLGGSSYEEEEEEEEGGGGGERKVGGPHPKYDEDAKRPYFTVDEAEARQDGYGDRTLGYQYDPEQLDLAENMVSQNDGSFISKKEWYVHuman PVRL2 with predicted signal sequence underlined (SEQ ID NO: 3)MARAAALLPSRSPPTPLLWPLLLLLLLETGAQDVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVEDEGNYTCEFATFPKGSVRGMTWLRVIAKPKNQAEAQKVTFSQDPTTVALCISKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVPSGRADGVTVTCKVEHESFEEPALIPVTLSVRYPPEVSISGYDDNWYLGRTDATLSCDVRSNPEPTGYDWSTTSGTFPTSAVAQGSQLVIHAVDSLFNTTFVCTVTNAVGMGRAEQVIFVRETPNTAGAGATGGIIGGIIAAIIATAVAATGILICRQQRKEQTLQGAEEDEDLEGPPSYKPPTPKAKLEAQEMPSQLFTLGASEHSPLKTPYFDAGASCTEQEMPRYHELPTLEERSGPLHPGATSLGSPIPVPPGPPAVEDVSLDLEDEEGEEEEEYLDKINPIYDALSYSSPSDSYQGKGFVMSRAMYVHuman PVRL3 with predicted signal sequence underlined (SEQ ID NO: 4)MARTLRPSPLCPGGGKAQLSSASLLGAGLLLQPPTPPPLLLLLFPLLLFSRLCGALAGPIIVEPHVTAVWGKNVSLKCLIEVNETITQISWEKIHGKSSQTVAVHHPQYGFSVQGEYQGRVLFKNYSLNDATITLHNIGFSDSGKYICKAVTFPLGNAQSSTTVTVLVEPTVSLIKGPDSLIDGGNETVAAICIAATGKPVAHIDWEGDLGEMESTTTSFPNETATIISQYKLFPTRFARGRRITCVVKHPALEKDIRYSFILDIQYAPEVSVTGYDGNWFVGRKGVNLKCNADANPPPFKSVWSRLDGQWPDGLLASDNTLHFVHPLTFNYSGVYICKVTNSLGQRSDQKVIYISDPPTTTTLQPTIQWHPSTADIEDLATEPKKLPFPLSTLATIKDDTIATIIASVVGGALFIVLVSVLAGIFCYRRRRTFRGDYFAKNYIPPSDMQKESQIDVLQQDELDSYPDSVKKENKNPVNNLIRKDYLEEPEKTQWNNVENLNRFERPMDYYEDLKMGMKFVSDEHYDENEDDLVSHVDGS VISRREWYVHuman PVRL4 with predicted signal sequence underlined (SEQ ID NO: 4)MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLVHuman TIGIT with predicted signal sequence underlined (SEQ ID NO: 6)MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFF TETGHuman CD96 with predicted signal sequence underlined (SEQ ID NO: 7)MEKKWKYCAVYYIIQIHFVKGVWEKTVNTEENVYATLGSDVNLTCQTQTVGFFVQMQWSKVTNKIDLIAVYHPQYGFYCAYGRPCESLVTFTETPENGSKWTLHLRNMSCSVSGRYECMLVLYPEGIQTKIYNLLIQTHVTADEWNSNHTIEIEINQTLEIPCFQNSSSKISSEFTYAWSVENSSTDSWVLLSKGIKEDNGTQETLISQNHLISNSTLLKDRVKLGTDYRLHLSPVQIFDDGRKFSCHIRVGPNKILRSSTTVKVFAKPEIPVIVENNSTDVLVERRFTCLLKNVFPKANITWFIDGSFLHDEKEGIYITNEERKGKDGFLELKSVLTRVHSNKPAQSDNLTIWCMALSPVPGNKVWNISSEKITFLLGSEISSTDPPLSVTESTLDTQPSPASSVSPARYPATSSVTLVDVSALRPNTTPQPSNSSMTTRGFNYPWTSSGTDTKKSVSRIPSETYSSSPSGAGSTLHDNVFTSTARAFSEVPTTANGSTKTNHVHITGIVVNKPKDGMSWPVIVAALLFCCMILFGLGVRKWCQYQKEIMERPPPFKPPPPPIKYTCIQEPNESDLPYHEMETLHuman CD226 with predicted signal sequence underlined (SEQ ID NO: 8)MDYPTLLLALLHVYRALCEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVAGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV PVR FamilyHuman PVR- ECD without predicted signal sequence (SEQ ID NO: 9)DVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGMSRN Human PVRL1- ECD without predicted signal sequence(SEQ ID NO: 10)QVVQVNDSMYGFIGTDVVLHCSFANPLPSVKITQVTWQKSTNGSKQNVAIYNPSMGVSVLAPYRERVEFLRPSFTDGTIRLSRLELEDEGVYICEFATFPTGNRESQLNLTVMAKPTNWIEGTQAVLRAKKGQDDKVLVATCTSANGKPPSVVSWETRLKGEAEYQEIRNPNGTVTVISRYRLVPSREAHQQSLACIVNYHMDRFKESLTLNVQYEPEVTIEGFDGNWYLQRMDVKLTCKADANPPATEYHWTTLNGSLPKGVEAQNRTLFFKGPINYSLAGTYICEATNPIGTRSGQVEVNITEFPYTPSPPEHGRRAGPVPTAHuman PVRL2- ECD without predicted signal sequence (SEQ ID NO: 11)QDVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVEDEGNYTCEFATFPKGSVRGMTWLRVIAKPKNQAEAQKVTFSQDPTTVALCISKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVPSGRADGVTVTCKVEHESFEEPALIPVTLSVRYPPEVSISGYDDNWYLGRTDATLSCDVRSNPEPTGYDWSTTSGTFPTSAVAQGSQLVIHAVDSLFNTTFVCTVTNAVGMGRAEQVIFVRETPNTAGAGATGGHuman PVRL3- ECD without predicted signal sequence (SEQ ID NO: 12)GPIIVEPHVTAVWGKNVSLKCLIEVNETITQISWEKIHGKSSQTVAVHHPQYGFSVQGEYQGRVLFKNYSLNDATITLHNIGFSDSGKYICKAVTFPLGNAQSSTTVTVLVEPTVSLIKGPDSLIDGGNETVAAICIAATGKPVAHIDWEGDLGEMESTTTSFPNETATIISQYKLFPTRFARGRRITCVVKHPALEKDIRYSFILDIQYAPEVSVTGYDGNWFVGRKGVNLKCNADANPPPFKSVWSRLDGQWPDGLLASDNTLHFVHPLTFNYSGVYICKVTNSLGQRSDQKVIYISDPPTTTTLQPTIQWHPSTADIEDLATEPKKLPFPLSTLATIKDDTIATHuman PVRL4- ECD without predicted signal sequence (SEQ ID NO: 13)GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSAS Human TIGIT- ECD without predicted signal sequence(SEQ ID NO: 14)MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVA Human CD96- ECD without predicted signal sequence(SEQ ID NO: 15)KTVNTEENVYATLGSDVNLTCQTQTVGFFVQMQWSKVTNKIDLIAVYHPQYGFYCAYGRPCESLVTFTETPENGSKWTLHLRNMSCSVSGRYECMLVLYPEGIQTKIYNLLIQTHVTADEWNSNHTIEIEINQTLEIPCFQNSSSKISSEFTYAWSVENSSTDSWVLLSKGIKEDNGTQETLISQNHLISNSTLLKDRVKLGTDYRLHLSPVQIFDDGRKFSCHIRVGPNKILRSSTTVKVFAKPEIPVIVENNSTDVLVERRFTCLLKNVFPKANITWFIDGSFLHDEKEGIYITNEERKGKDGFLELKSVLTRVHSNKPAQSDNLTIWCMALSPVPGNKVWNISSEKITFLLGSEISSTDPPLSVTESTLDTQPSPASSVSPARYPATSSVTLVDVSALRPNTTPQPSNSSMTTRGFNYPWTSSGTDTKKSVSRIPSETYSSSPSGAGSTLHDNVFTSTARAFSEVPTTANGSTKTNHVHITGIVVNKPKDGMS Human CD226- ECD without predicted signal sequence(SEQ ID NO: 16)EEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVAHuman PVR- N-terminal IgV domain (SEQ ID NO: 17)DVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAVariant 1 Human PVR- N-terminal IgV domain (SEQ ID NO: 18)DVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLXWXRHGEXXXMAVFHQXXGXXYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWL X = any amino acidVariant 2 Human PVR- N-terminal IgV domain (SEQ ID NO: 19)DVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGENGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLVariant 3 Human PVR- N-terminal IgV domain (SEQ ID NO: 20)DVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTKGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLVariant 4 Human PVR- N-terminal IgV domain (SEQ ID NO: 21)DVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGENGSMAVFHQTKGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLHuman PVRL1- N-terminal IgV domain (SEQ ID NO: 22)QVVQVNDSMYGFIGTDVVLHCSFANPLPSVKITQVTWQKSTNGSKQNVAIYNPSMGVSVLAPYRERVEFLRPSFTDGTIRLSRLELEDEGVYICEFATFPTGNRESQLNLTVMAHuman PVRL2- N-terminal IgV domain (SEQ ID NO: 23)DVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVEDEGNYTCEFATFPKGSVRG MTWLRVIAHuman PVRL3- N-terminal IgV domain (SEQ ID NO: 24)GPIIVEPHVTAVWGKNVSLKCLIEVNETITQISWEKIHGKSSQTVAVHHPQYGFSVQGEYQGRVLFKNYSLNDATITLHNIGFSDSGKYICKAVTFPLGNAQSSTTVTVLVHuman PVRL4- N-terminal IgV domain (SEQ ID NO: 25)GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPHuman IgG₁ Fc region (SEQ ID NO: 26)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG₁ Fc region(SEQ ID NO: 27)KSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG₁ Fc region(SEQ ID NO: 28)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG2 Fc region (SEQ ID NO: 29)CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG2 Fc region (13B chain) (SEQ ID NO: 30)CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG2 Fc region (13A chain) (SEQ ID NO: 31)CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FLAG Tag (SEQ ID NO: 32)DYKDDDDK Linker (SEQ ID NO: 33) ESGGGGVT Linker (SEQ ID NO: 34)LESGGGGVT Linker (SEQ ID NO: 35) GRAQVT Linker (SEQ ID NO: 36) WRAQVTLinker (SEQ ID NO: 37) ARGRAQVTVariant human PVRL2- N-terminal IgV domain (SEQ ID NO: 38)DVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVXWXRPDAPANXXXVAAFHPXXGXXFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVEDEGNYTCEFATFPKGSVRG MTWLRVIAX = any amino acid Human IgG1 Heavy chain constant region(SEQ ID NO: 39)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Heavy chain constant region(SEQ ID NO: 40)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG3 Heavy chain constant region(SEQ ID NO: 41)ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK Human IgG4 Heavy chain constant region (SEQ ID NO: 42)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Human IgG₂ Fc region (SEQ ID NO: 43)TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG₂ Fc region variant (SEQ ID NO: 44)TKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG₂ Fc region (Variant 13A) (SEQ ID NO: 45)TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG₂ Fc region variant (Variant 13A) (SEQ ID NO: 46)TKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG₂ Fc region (Variant 13B) (SEQ ID NO: 47)TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG₂ Fc region variant (Variant 13B) (SEQ ID NO: 48)TKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

1-96. (canceled)
 97. A method of increasing an immune response in asubject comprising: administering to the subject a therapeuticallyeffective amount of a soluble receptor comprising a PVR variant, whereinthe soluble receptor: (i) inhibits the interaction between TIGIT andPVR; (ii) inhibits the interaction between CD96 and PVR; (iii) inhibitsthe interaction between TIGIT and PVR and inhibits the interactionbetween CD96 and PVR; or (iv) inhibits the interaction between TIGIT andPVR, inhibits the interaction between CD96 and PVR, and does not inhibitthe interaction between CD226 and PVR.
 98. A method of inhibiting tumorgrowth in a subject comprising: administering to the subject atherapeutically effective amount of a soluble receptor comprising a PVRvariant, wherein the soluble receptor: (i) inhibits the interactionbetween TIGIT and PVR; (ii) inhibits the interaction between CD96 andPVR; (iii) inhibits the interaction between TIGIT and PVR and inhibitsthe interaction between CD96 and PVR; or (iv) inhibits the interactionbetween TIGIT and PVR, inhibits the interaction between CD96 and PVR,and does not inhibit the interaction between CD226 and PVR.
 99. Themethod of claim 97, wherein the PVR variant comprises the amino acidsequence of wild-type PVR (SEQ ID NO:1), except for one or more aminoacid substitutions of amino acid residues selected from the groupconsisting of 65, 67, 72, 73, 74, 81, 82, 84, and 85 of wild-type PVR(SEQ ID NO:1).
 100. The method of claim 98, wherein the PVR variantcomprises the amino acid sequence of wild-type PVR (SEQ ID NO:1), exceptfor one or more amino acid substitutions of amino acid residues selectedfrom the group consisting of 65, 67, 72, 73, 74, 81, 82, 84, and 85 ofwild-type PVR (SEQ ID NO:1).
 101. The method of claim 99, wherein theone or more amino acid substitutions within the PVR variant comprisesubstitutions in one or more amino acids: (a) corresponding to aminoacid 72 of wild-type PVR (SEQ ID NO:1); (b) corresponding to amino acid82 of wild-type PVR (SEQ ID NO:1); or (c) corresponding to amino acid 72and amino acid 82 of wild-type PVR (SEQ ID NO:1).
 102. The method ofclaim 100, wherein the one or more amino acid substitutions within thePVR variant comprise substitutions in one or more amino acids: (a)corresponding to amino acid 72 of wild-type PVR (SEQ ID NO:1); (b)corresponding to amino acid 82 of wild-type PVR (SEQ ID NO:1); or (c)corresponding to amino acid 72 and amino acid 82 of wild-type PVR (SEQID NO:1).
 103. The method of claim 99, wherein the PVR variant comprisesan amino acid sequence selected from the group consisting of: SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.
 104. The method ofclaim 100, wherein the PVR variant comprises an amino acid sequenceselected from the group consisting of: SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20, and SEQ ID NO:21.
 105. The method of claim 97, wherein theimmune response is directed to a tumor cell or cancer.
 106. The methodof claim 97, further comprising the administration of at least oneadditional therapeutic agent.
 107. The method of claim 106, wherein theadditional therapeutic agent is an immune response stimulating agent, achemotherapeutic agent, a small molecule agent, and/or an antibody. 108.The method of claim 107, wherein the additional therapeutic agent is animmune response stimulating agent comprising an antibody that blocksimmunosuppressive functions.
 109. The method of claim 98, furthercomprising the administration of at least one additional therapeuticagent.
 110. The method of claim 109, wherein the additional therapeuticagent is an immune response stimulating agent, a chemotherapeutic agent,a small molecule agent, and/or an antibody.
 111. The method of claim110, wherein the additional therapeutic agent is an immune responsestimulating agent comprising an antibody that blocks immunosuppressivefunctions.
 112. A cell comprising or producing a polypeptide, whereinthe polypeptide comprises a poliovirus receptor (PVR) variant, whereinthe PVR variant comprises the amino acid sequence of wild-type PVR (SEQID NO:1), except for one or more amino acid substitutions of amino acidresidues selected from the group consisting of 65, 67, 72, 73, 74, 81,82, 84, and 85 of wild-type PVR (SEQ ID NO:1).
 113. A polynucleotideencoding the polypeptide produced by the cell of claim
 112. 114. Avector comprising the polynucleotide of claim
 113. 115. A kit comprisingthe polynucleotide of claim 113.